Stabilized aptamers to platelet derived growth factor and their use as oncology therapeutics

ABSTRACT

Materials and methods are provided for producing and using aptamers useful as oncology therapeutics capable of binding to PDGF, PDGF isoforms, PDGF receptor, VEGF, and VEGF receptor or any combination thereof with great affinity and specificity. The compositions of the present invention are particularly useful in solid tumor therapy and can be used alone or in combination with known cytotoxic agents for the treatment of solid tumors. Also disclosed are aptamers having one or more CpG motifs embedded therein or appended thereto.

REFERENCE TO RELATED APPLICATIONS

[0001] This non-provisional patent application is a continuation-in-partof U.S. Ser. No. 10/762,915, filed Jan. 21, 2004, which claims priorityto U.S. Ser. No. 60/441,357, filed on Jan. 21, 2003; U.S. Ser. No.60/463,095, filed on Apr. 15, 2003; U.S. Ser. No. 60/464,179, filed onApr. 21, 2003; U.S. Ser. No. 60/465,055, filed Apr. 23, 2003; U.S. Ser.No. 60/469,628, filed May 8, 2003; U.S. Ser. No. 60/474,680, filed May29, 2003; U.S. Ser. No. 60/491,019, filed Jul. 29, 2003; U.S. Ser. No.60/512,071, filed Oct. 17, 2003; U.S. Ser. No. 60/537,201, filed Jan.16, 2004; and U.S. Ser. No. 60/537,045, filed Jan. 16, 2004; is acontinuation-in-part of U.S. Ser. No. 10/718,833, filed Nov. 21, 2003,which claims priority to U.S. Ser. No. 60/428,102, filed Nov. 21, 2002and U.S. Ser. No. 60/469,628, filed on May 8, 2003; and is acontinuation in part of U.S. Ser. No. 10/______, entitled “Aptamer ToxinMolecules and Methods for Using Same”, filed on Apr. 15, 2004; and thispatent application claims priority under 35 U.S.C. § 119(e) to thefollowing provisional applications: U.S. Ser. No. 60/464,239, filed Apr.21, 2003; U.S. Ser. No. 60/465,053, filed Apr. 23, 2003; U.S. Ser. No.60/469,628, filed May 8, 2003; U.S. Ser. No. 60/474,133, filed May 29,2003; U.S. Ser. No. 60/474,680, filed May 29, 2003; U.S. Ser. No.60/486,580, filed Jul. 11, 2003; U.S. Ser. No. 60/489,810, filed Jul.23, 2003; U.S. Ser. No. 60/491,019, filed Jul. 29, 2003; U.S. Ser. No.60/503,596, filed Sep. 16, 2003; each of which is herein incorporated byreference in its entirety. The present non-provisional patentapplication is related to U.S. Ser. No. 60/461,966, filed Apr. 10, 2003,now abandoned, and to U.S. Ser. No. 60/462,779, filed Apr. 14, 2003, nowabandoned, each of which is herein incorporated by reference.

FIELD OF THE INVENTION

[0002] The invention relates generally to the field of nucleic acids andmore particularly to aptamers capable of binding to platelet derivedgrowth factor (“PDGF”) useful as therapeutics in oncology and/or otherdiseases or disorders in which PDGF has been implicated. The inventionfurther relates to materials and methods for the administration ofaptamers capable of binding to platelet derived growth factor.

BACKGROUND OF THE INVENTION

[0003] Aptamers are nucleic acid molecules having specific bindingaffinity to molecules through interactions other than classicWatson-Crick base pairing.

[0004] Aptamers, like peptides generated by phage display or monoclonalantibodies (MAbs), are capable of specifically binding to selectedtargets and, through binding, block their targets' ability to function.Created by an in vitro selection process from pools of random sequenceoligonucleotides (FIG. 1), aptamers have been generated for over 100proteins including growth factors, transcription factors, enzymes,immunoglobulins, and receptors. A typical aptamer is 10-15 kDa in size(30-45 nucleotides), binds its target with sub-nanomolar affinity, anddiscriminates against closely related targets (e.g., will typically notbind other proteins from the same gene family). A series of structuralstudies have shown that aptamers are capable of using the same types ofbinding interactions (hydrogen bonding, electrostatic complementarity,hydrophobic contacts, steric exclusion, etc.) that drive affinity andspecificity in antibody-antigen complexes.

[0005] Aptamers have a number of desirable characteristics for use astherapeutics (and diagnostics) including high specificity and affinity,biological efficacy, and excellent pharmacokinetic properties. Inaddition, they offer specific competitive advantages over antibodies andother protein biologics, for example:

[0006] 1) Speed and control. Aptamers are produced by an entirely invitro process, allowing for the rapid generation of initial(therapeutic) leads. In vitro selection allows the specificity andaffinity of the aptamer to be tightly controlled and allows thegeneration of leads against both toxic and non-immunogenic targets.

[0007] 2) Toxicity and Immunogenicity. Aptamers as a class havedemonstrated little or no toxicity or immunogenicity. In chronic dosingof rats or woodchucks with high levels of aptamer (10 mg/kg daily for 90days), no toxicity is observed by any clinical, cellular, or biochemicalmeasure. Whereas the efficacy of many monoclonal antibodies can beseverely limited by immune response to antibodies themselves, it isextremely difficult to elicit antibodies to aptamers (most likelybecause aptamers cannot be presented by T-cells via the MHC and theimmune response is generally trained not to recognize nucleic acidfragments).

[0008] 3) Administration. Whereas all currently approved antibodytherapeutics are administered by intravenous infusion (typically over2-4 hours), aptamers can be administered by subcutaneous injection. Thisdifference is primarily due to the comparatively low solubility and thuslarge volumes necessary for most therapeutic MAbs. With good solubility(>150 mg/ml) and comparatively low molecular weight (aptamer: 10-50 kDa;antibody: 150 kDa), a weekly dose of aptamer may be delivered byinjection in a volume of less than 0.5 ml. Aptamer bioavailability viasubcutaneous administration is >80% in monkey studies (Tucker et al., J.Chromatography B. 732: 203-212, 1999). In addition, the small size ofaptamers allows them to penetrate into areas of conformationalconstrictions that do not allow for antibodies or antibody fragments topenetrate, presenting yet another advantage of aptamer-basedtherapeutics or prophylaxis.

[0009]4) Scalability and cost. Therapeutic aptamers are chemicallysynthesized and consequently can be readily scaled as needed to meetproduction demand. Whereas difficulties in scaling production arecurrently limiting the availability of some biologics and the capitalcost of a large-scale protein production plant is enormous, a singlelarge-scale synthesizer can produce upwards of 100 kg oligonucleotideper year and requires a relatively modest initial investment. Thecurrent cost of goods for aptamer synthesis at the kilogram scale isestimated at $500/g, comparable to that for highly optimized antibodies.Continuing improvements in process development are expected to lower thecost of goods to <$100/g in five years.

[0010] 5) Stability. Therapeutic aptamers are chemically robust. Theyare intrinsically adapted to regain activity following exposure to heat,denaturants, etc. and can be stored for extended periods (>1 yr) at roomtemperature as lyophilized powders. In contrast, antibodies must bestored refrigerated.

[0011] Interstitial Fluid Pressure

[0012] The three most common types of cancer treatment are surgicalremoval of cancerous tissue, radiotherapy to obliterate canceroustissue, and chemotherapy. These treatments are aimed at removing thecancerous tissues or cells or destroying them in the body withtherapeutics or other agents. Chemotherapy remains a major treatmentmodality for solid tumors. To potentially reduce toxic side effects andto achieve higher efficacy of chemotherapeutic drugs, strategies toimprove distribution of drugs between normal tissues and tumors arehighly desirable.

[0013] A major obstacle in the treatment of solid tumors is the limiteduptake of therapeutic agents into tumor tissue. Elevated interstitialfluid pressure (“IFP”) is one of the physiologically distinctiveproperties of solid tumors that differ from healthy connective tissueand is considered to be the main obstacle limiting free diffusion oftherapeutics into solid tumors. PDGF receptors, particularly PDGF-β,have been implicated in the regulation of IFP. As a tumor enters ahyperproliferative state, blood supplying oxygen and other nutrientscannot keep up with the tumors' demands and a state of hypoxia results.Hypoxia triggers an “angiogenic switch” which will up-regulate theexpression of several factors including VEGF and PDGF which in turnserve to initiate angiogenesis. However, the angiogenesis that resultsforms an abnormal tumor vasculature. The tumor vasculature becomesimpaired to the point of being unable to adequately drain excess fluidfrom the interstitium and fluid accumulation distends the elasticinterstitial matrix causing an increase in pressure. When pressureexceeds capillary wall resistance, compression occurs and blood flowresistance increases.

[0014] This property of most solid tumors—tumor interstitialhypertension or increased IFP—has been suggested as a potential targetfor efforts to increase tumor drug uptake (Jain et al., (1987) CancerRes., 47:3039-3051). Increased IFP acts as a barrier for tumortransvascular transport (Jain et al. (1997), Adv. Drug Deliv. Rev.26:71-90). Lessening of tumor IFP, or modulation of microvascularpressure, has been shown to increase transvascular transport oftumor-targeting antibodies or low-molecular weight tracer compounds(Pietras et al., (2001), Cancer Res., 61, 2929-2934). The etiology ofinterstitial hypertension in tumors is poorly understood. One proposedtheory is that the lack of lymphatic vessels in tumors is a contributingfactor to the increased tumor IFP (Jain et al., (1987), Cancer Res.,47:3039-3051). Another proposed theory is that the microvasculature andthe supporting stroma compartment are likely to be importantdeterminants for tumor IFP (Pietras et al., (2002) Cancer Res., 62:5476-5484). Accumulating evidence points toward the transmembrane PDGFβ-receptor tyrosine kinase as a potential target for pharmacologicaltherapeutics to modulate tumor interstitial hypertension. Among otherpotential targets are growth factors that bind to the PDGF β-receptor.

[0015] PDGF Mediated Cancer

[0016] In addition to IFP and the difficulty of penetrating tumors withtherapeutics, another obstacle in cancer treatment are mutations incertain forms of cancer by PDGF mediated cancer leading to constitutiveexpression of PDGF. These mutations drive abnormal proliferation ofcells which results in the various forms of cancer as shown in FIG. 3(Pietras et al., (2001), Cancer Res., 61, 2929-2934). A gene mutationresults in amplification of PDGF α-receptors in high gradeglioblastomas. In chronic myelomonocytic leukemia (CMML), constitutiveactivation of PDGF-β receptors results from a mutation which causes thefusion of β-receptors with proteins other than PDGF (Golub et al.,(1994) Cell 77, 307-316, Magnusson et al., (2001) Blood 100, 623-626).Constitutive activation of PDGF-α-receptor due to activating pointmutations has also been identified in patients with gastrointestinalstromal tumors (GIST) (Heinreich et al.,(2003) Science 299, 708-710).Dermatofibrosarcoma protuberans (DFSP) is associated with constitutiveproduction of fusion proteins which are processed to PDGF-BB (O'Brian etal., (1998) Gene Chrom. Cancer 23, 187-193; Shimiziu et al. (1999)Cancer Res. 59, 3719-3723; Simon et al. (1997) Nat. Genet., 15, 95-98).In addition to the constitutive activation of PDGF ligand and/orreceptor due to mutations, up regulation has been shown in soft tissuesarcomas and gliomas (Ostman and Heldin, (2001), Adv. Cancer Res. 80,1-38).

[0017] PDGF

[0018] Growth factors are substances that have a cell-proliferativeeffect on cells or tissues. Any given growth factor may have more thanone receptor or induce cell proliferation in more than one cell line ortissue. PDGF belongs to the cysteine-knot growth factor family and wasoriginally isolated from platelets for promoting cellular mitogenic andmigratory activity. PDGF is a strong mitogen and has a pivotal role inregulation of normal cell proliferation such as fibroblasts, smoothmuscle cells, neuroglial cells and connective-tissue cells. In addition,PDGF mediates pathological cell growth such as in proliferativedisorders, and also plays a role in angiogenesis. Another growth factorinvolved in tumor angiogenesis is vascular endothelial growth factor(VEGF).

[0019] Four PDGF polypeptide chains have been identified which arecurrently known to make up five dimeric PDGF isoforms: PDGF-AA, -BB,-CC, -DD, and -AB. The most abundant species are PDGF AB and BB. PDGFisoforms bind to α and β tyrosine kinase receptors. PDGF receptors areexpressed by many different cell types within tumors. The binding ofPDGF isoforms to their cognate receptors induces the dimerization andsubsequent phosphorylation of specific residues in the intracellulartyrosine kinase domain of the receptors and activation of the signalingpathway. PDGF isoforms -AA, -BB, -CC, and -AB induce PDGF α-receptordimerization. PDGF-BB and PDGF-DD activate PDGF β receptor dimerization.All isoforms of PDGF except PDGF-AA activate both α and β receptors incells which co-express both receptor types (FIG. 2). Because they arepotent mitogens, PDGF isoforms have been targeted for proliferativedisease therapeutics development, such as cancer, diabetic retinopathy,glomerulonephritis, and restenosis.

[0020] PDGF, which is secreted by endothelial cells, acts as directmitogen for fibroblasts, recruits pericytes and stimulates vascularsmooth muscle cells. Many solid tumors display paracrine signaling ofPDGF in the tumor stroma. PDGF is known to up-regulate synthesis ofcollagen and to mediate interactions of anchor proteins such asintegrins with extracellular matrix (ECM) components. PDGF interactionsbetween connective tissue, ECM and intracellular actin filament systemscause increased tensile strength which contributes to high IFP. High IFPis localized to the site of tumor and is associated with poor prognosisin human cancers as it increases with tumor size and severity and thegrade of malignancy. The role of PDGF signaling in control of IFP andthe up-regulated expression in various solid tumors, has promptedinvestigation into whether the inhibition of PDGF signaling can decreaseIFP and thereby increase drug uptake into solid tumors. Previous workhas demonstrated that inhibition of PDGF signaling with small moleculereceptor antagonists and a PDGF specific aptamer decreases interstitialfluid pressure and increases the uptake of chemotherapeutics into solidtumors (Pietras et al., (2001), Cancer Res., 61: 2929-2934).

[0021] Accordingly, it would be beneficial to have novel materials andmethods in oncology therapy to reduce tumor IFP, decrease tumorangiogenesis, and reduce the deleterious effects of mutation by theconstitutive expression of PDGF. The present invention providesmaterials and methods to meet these and other needs.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 is a schematic representation of the in vitro aptamerselection (SELEX™) process from pools of random sequenceoligonucleotides.

[0023]FIG. 2 is a schematic of isoforms AA, BB, CC, DD, and AB of PDGFand cognate receptors.

[0024]FIG. 3 is a schematic of gene mutations that give rise toconstitutive PDGF receptor signaling in cancer cells found inglioblastomas, chronic myelomonocytic leukemia (CMML),dermatofibrosarcoma protuberans (DFSP), gastrointestinal stromal tumors(GIST), and other soft tissue sarcomas.

[0025]FIG. 4 is a schematic of the transport of cytotoxins across tumorvasculature with and without PDGF antagonists by the methods of thepresent invention.

[0026]FIG. 5A is a plot of an ion-exchange HPLC analysis of ARC 127(5′-[40K PEG]-(SEQ ID NO:1)-HEG-(SEQ ID NO:2)-HEG-(SEQ ID NO:3)-3′dT-3′) freshly synthesized and stored at −20° C. for two years shows;FIG. 5B is a bar-graph of 3T3 cell proliferation assay results forARC126 (5′-(SEQ ID NO:1)-HEG-(SEQ ID NO:2)-HEG-(SEQ ID NO:3)-3′ dT-3′)and ARC127 newly synthesized and after storage at −20° C. for 2 years.

[0027]FIG. 6A is a schematic of the sequence and secondary structure of2′-fluoro-containing PDGF aptamer composition of ARC126; FIG. 6B is aschematic of the sequence and secondary structure of ARC126 variantsARC513 (5′-(SEQ ID NO:86)-PEG-(SEQ ID NO:57)-PEG-(SEQ ID NO:87)-3′dT-3′), ARC514 (5′-(SEQ ID NO:86)-PEG-(SEQ ID NO:57)-PEG-(SEQ IDNO:88)-3′ dT-3′), ARC515 (5′-(SEQ ID NO:86)-PEG-(SEQ ID NO:57)-PEG-(SEQID NO:89)-3′ dT-3′), and ARC516 (5′-(SEQ ID NO:86)-PEG-(SEQ IDNO:57)-PEG-(SEQ ID NO:90)-3′ dT-3′).

[0028]FIG. 7A is a plot of competition binding assay results for ARC126and composition variants ARC128 (5′-(SEQ ID NO:4)-HEG-(SEQ IDNO:5)-HEG-(SEQ ID NO:6)-3′), ARC513, ARC514, ARC515, ARC516; FIG. 7B isa plot of in vitro 3T3 cell-based proliferation assay data showing theactivity of some composition variants of ARC126.

[0029]FIG. 8A is a plot of a competition binding assay for ARC126 andtwo variants that are 5′ conjugated to 30 kD (ARC308, 5′-[30K PEG]-(SEQID NO:1)-HEG-(SEQ ID NO:2)-HEG-(SEQ ID NO:3)-3′ dT-3′) and 40 kD(ARC127) PEG groups; FIG. 8B is a plot of in vitro 3T3 cell-basedproliferation assay data for ARC126 as a function of 5′ PEG groupconjugation (ARC126+30 kD=ARC308, and ARC126+40 kD PEG=ARC127).

[0030]FIG. 9A is a plot of a pharmacokinetic profile of 5′ conjugates ofARC126 after IV administration at 10 mg/kg in mice; FIG. 9B is a plot ofthe in vivo pharmacokinetic profile of ARC127 (ARC126+40 kD PEG) afterintravenous (IV), intraperitoneal (IP), and subcutaneous (SC)administration at a dose level of 10 mg/kg in mice; FIG. 9C is a plot ofthe bioactivity profile of ARC126+40 kD PEG (i.e., ARC127) afterintravenous (IV) administration at a dose level of 10 mg/kg in mice.

[0031]FIG. 10A is a plot of a competition binding assay showing thatARC126 binds to PDGF-BB with a K_(d) of approximately 100 pM, andPDGF-AB with a K_(d) of approximately 100 pM, but does not bind toPDGF-AA; FIG. 11B is a plot of a competition binding assay showing thatARC126 binds to human, rat and mouse PDGF-BB with an equal affinity ofapproximately 100 pM.

[0032]FIG. 11A is a plot of the results of a 3T3 cell proliferationassay showing that ARC127 inhibits 3T3 cell proliferation better thanPDGF Neutralizing antibody; FIG. 11B is a plot of the results of a 3T3cell proliferation assay showing that ARC127 inhibits 3T3 cellproliferation better than known tyrosine kinase inhibitors tested.

[0033]FIG. 12 is a plot of a cell viability assay results showing thatARC127 alone does not have any toxic effect on 3T3 cells.

[0034]FIG. 13 is a plot of cell migration assay data performed in 96well format using QCM Chemotaxis 96 Well Migration Assay (#ECM 510)(Chemicon, Temecula, Calif.) showing that the migration signal observedincreases as a function of the number of cells plated or increasing PDGFconcentration.

[0035]FIG. 14 is a plot of the results of a PDGF-driven Elk Luciferaseassay showing the ARC127 displays an IC₅₀ of 2 nM.

[0036]FIG. 15A is a plot of tumor diameter versus time in Nu/Nu nudemice under a GLEEVEC™/irinotecan dose optimization study in HT29 coloncancer xenograft model; FIG. 15B is a of plot tumor diameter versus timein Nu/Nu nude mice under ARC127/irinotecan study in LS174T colon cancerxenograft model.

[0037]FIG. 16 is a plot of tumor diameter versus time in Nu/Nu nude miceunder irinotecan, and GLEEVEC/ARC308 dosing regimens in an efficacystudy in LS174T colon cancer xenotransplant model.

[0038]FIG. 17A is a schematic of the sequence and secondary structure ofan aptamer that binds to VEGF but not PDGF-ARC245 (SEQ ID NO:7); FIG.17B is a schematic of the sequence and secondary structure of an aptamerthat binds to PDGF but not VEGF—referred to herein as ARC126.

[0039]FIG. 18 is a schematic of the sequence and secondary structure ofa bivalent aptamer that binds to PDGF and VEGF (sequence TK.131.012.A,SEQ ID NO:9); FIG. 18B is a schematic of the sequence and secondarystructure of a second bivalent aptamer that binds to PDGF and VEGF(sequence TK.131.012B, SEQ ID NO:10).

[0040]FIG. 19 is a plot of dot-blot assay binding data for theconstituent aptamers and the multivalent aptamers to PDGF BB (Panel 1)and VEGF (Panel 2).

[0041]FIG. 20 is a schematic of the sequence and secondary structures(absent an indication of the phosphorothioate bonds) of PDGF aptamershaving CpG islands or motifs incorporated or embedded therein.

[0042]FIG. 21A is a plot of the results of an IL-6 ELISA assay measuringIL-6 release in TIB cells using known immunostimulatory ODN's aspositive controls, and aptamers which contain no CpG islands as negativecontrols in the assay; FIG. 21B is a plot of the results of an IL-6ELISA assay measuring IL-6 release in TIB cells using the ISS ODN andshortened versions of the ISS ODN; FIG. 21C is a plot of the results ofan IL-6 ELISA assay measuring IL-6 release in TIB cells using PDGFaptamers in which CpG motifs have been incorporated; FIG. 21D is a plotof the results of an IL-6 ELISA assay measuring IL-6 release in TIBcells using additional PDGF aptamers in which CpG motifs have beenincorporated. FIG. 21E is a plot of the results of an TNFa ELISA assaymeasuring TNFA release in TIB cells using the same PDGF aptamers as inFIG. 21D in which CpG motifs have been incorporated.

[0043]FIG. 22 is an illustration depicting various strategies forsynthesis of high molecular weight PEG-nucleic acid conjugates.

[0044]FIGS. 23A-23B are graphs depicting the dot blot binding analysisfor clone RNA transcripts.

[0045]FIG. 24 is a graph depicting the inhibitory effect of variousPDGF-AA aptamers of the invention (ARX33P1.D2, ARX33P1.E5, andARX33P1.E10) on PDGF-AA induced 3T3 cell proliferation.

[0046]FIG. 25 is a graph depicting the effect of PDGF-AA aptamers onPDGF-BB induced 3T3 cell proliferation.

SUMMARY OF THE INVENTION

[0047] The present invention provides materials and methods for thetreatment of cancer, solid tumor cancers in particular, by theadministration to patients of therapeutically effective amounts ofaptamers or aptamer compositions capable of binding with great affinityand specificity to platelet derived growth factor, vascular endothelialgrowth factor, their isoforms, their receptors, or any combinationthereof, thus inhibiting the bound ligand's biological role in canceretiology. The aptamers of the present invention may be used with knownchemotherapeutic cytotoxic agents and may include one or more CpG motifsembedded therein or appended thereto.

[0048] The present invention provides aptamers that bind to PDGF. In oneembodiment, the aptamers that bind to PDGF include a nucleic acidsequence selected from SEQ ID NO:1 to SEQ ID NO:3, SEQ ID NO:9 to SEQ IDNO:38, SEQ ID NO:50, SEQ ID NO:54 to SEQ ID NO:90, and SEQ ID NO:94 toSEQ ID NO:99. In one embodiment, the oligonucleotide sequence of theaptamer contains less than seven nucleotides having a 2′ fluorosubstituent.

[0049] The invention also provides aptamers that include a firstsequence that binds to a first target and a second sequence that bindsto a second target. In one embodiment, the first target is PDGF,PDGF-isoforms, or PDGF receptor, and the second target is VEGF or VEGFreceptor. The PDGF isoforms are, for example, PDGF BB, PDGF AB, PDGF CC,and PDGF DD. In one embodiment, the aptamers that bind to PDGF include anucleic acid sequence selected from SEQ ID NO:1 to SEQ ID NO:3, SEQ IDNO:9 to SEQ ID NO:38, SEQ ID NO:50, SEQ ID NO:54 to SEQ ID NO:90, andSEQ ID NO:94 to SEQ ID NO:99.

[0050] In one embodiment, the first target does not, upon binding of theaptamer, stimulate an immune response, and moreover, the second targetdoes, upon binding of the aptamer, stimulate an immune response. In oneembodiment, the second target is a toll-like receptor. In anotherembodiment, the second sequence is an immunostimulatory sequence. In oneembodiment, the immunostimulatory sequence is a CpG motif.

[0051] In one embodiment, the first sequence is capable of binding toone of the following targets: PDGF, IgE, IgE Fcε R1, PSMA, CD22,TNF-alpha, CTLA4, PD-1, PD-L1, PD-L2, FcRIIB, BTLA, TIM-3, CD11c, BAFF,B7-X, CD19, CD20, CD25, and CD33. In one embodiment, the first sequenceis capable of binding to PDGF.

[0052] The present invention also provides compositions that contain aPDGF-binding aptamer of the invention and a pharmaceutically acceptablecarrier.

[0053] In one embodiment, the compositions of the invention include aPDGF-binding aptamer of the invention, a cytotoxic agent and apharmaceutically acceptable carrier. In one embodiment, the cytotoxicagent belongs to a class of cytotoxic agents such as tubulinstabilizers, tubulin destabilizers, anti-metabolites, purine synthesisinhibitors, nucleoside analogs, DNA alkylating agents, DNA modifyingagents, and vascular disrupting agents. In one embodiment, the cytotoxicagent is used alone or in combinations with one or more cytotoxic agentsselected from the group consisting of calicheamycin, doxorubicin, taxol,methotrexate, gemcitabine, cytarabine, vinblastin, daunorubicin,docetaxel, irinotecan, epothilone B, epothilone D, cisplatin,carboplatin, and 5-fluoro-U.

[0054] The invention also provides compositions that include aPDGF-binding aptamer of the invention, a VEGF-binding aptamer and apharmaceutically acceptable carrier. In one embodiment, thesecompositions also include a cytotoxic agent. Suitable cytotoxic agentsinclude agents belonging to a class of cytotoxic agents selected fromthe group consisting of tubulin stabilizers, tubulin destabilizers,anti-metabolites, purine synthesis inhibitors, nucleoside analogs, DNAalkylating agents, DNA modifying agents, and vascular disrupting agents.The cytotoxic agent is used alone or in combinations of one or morecytotoxic agents selected from the group consisting of calicheamycin,doxorubicin, taxol, methotrexate, gemcitabine, cytarabine, vinblastin,daunorubicin, docetaxel, irinotecan, epothilone B, epothilone D,cisplatin, carboplatin, and 5-fluoro-U.

[0055] The invention also provides methods of treating cancer byadministering a therapeutically effective amount of a PDGF-bindingaptamer of the invention. The invention also provides methods oftreating cancer by administering a therapeutically effective amount of acomposition of the invention.

[0056] In one embodiment, the cancer or tumor is PDGF mediated cancer ortumor. In one embodiment, the PDGF mediated cancer or tumor is aglioblastoma, chronic myelomonocytic leukemia, a dermafibrosarcomaprotuberan, a gastrointestinal stromal tumor or a soft tissue sarcoma.

[0057] In one embodiment, the composition includes a cytotoxic agentthat belongs to a class of cytotoxic agents selected from the groupconsisting of tubulin stabilizers, tubulin destabilizers,anti-metabolites, purine synthesis inhibitors, nucleoside analogs, DNAalkylating agents, DNA modifying agents, and vascular disrupting agents.In one embodiment, the cytotoxic agent is used alone or in combinationsof one or more cytotoxic agents such as calicheamycin, doxorubicin,taxol, methotrexate, gemcitabine, cytarabine, vinblastin, daunorubicin,docetaxel, irinotecan, epothilone B, epothilone D, cisplatin,carboplatin, and 5-fluoro-U.

[0058] The invention also provides methods of inhibiting growth of asolid tumor by administering a therapeutically effective amount of aPDGF-binding aptamer of the invention. The invention also providesmethods of inhibiting growth of a solid tumor by administering atherapeutically effective amount of a composition of the invention.

[0059] In one embodiment, the cancer or tumor is PDGF mediated cancer ortumor. In one embodiment, the PDGF mediated cancer or tumor is aglioblastoma, chronic myelomonocytic leukemia, a dermafibrosarcomaprotuberan, a gastrointestinal stromal tumor or a soft tissue sarcoma.

[0060] In one embodiment, the composition includes a cytotoxic agentbelonging to a class of cytotoxic agents selected from the groupconsisting of tubulin stabilizers, tubulin destabilizers,anti-metabolites, purine synthesis inhibitors, nucleoside analogs, DNAalkylating agents, DNA modifying agents, and vascular disrupting agents.In one embodiment, the cytotoxic agent is used alone or in combinationsof one or more cytotoxic agents such as calicheamycin, doxorubicin,taxol, methotrexate, gemcitabine, cytarabine, vinblastin, daunorubicin,docetaxel, irinotecan, epothilone B, epothilone D, cisplatin,carboplatin, and 5-fluoro-U.

[0061] The invention also provides methods of reducing IFP in a solidtumor by administering a therapeutically effective amount of aPDGF-binding aptamer of the invention. The invention also providesmethods of reducing IFP in a solid tumor by administering atherapeutically effective amount of a composition of the invention.

[0062] In one embodiment, the cancer or tumor is PDGF mediated cancer ortumor. In one embodiment, the PDGF mediated cancer or tumor is aglioblastoma, chronic myelomonocytic leukemia, a dermafibrosarcomaprotuberan, a gastrointestinal stromal tumor or a soft tissue sarcoma.

[0063] In one embodiment, the composition includes a cytotoxic agentbelonging to a class of cytotoxic agents selected from the groupconsisting of tubulin stabilizers, tubulin destabilizers,anti-metabolites, purine synthesis inhibitors, nucleoside analogs, DNAalkylating agents, DNA modifying agents, and vascular disrupting agents.In one embodiment, the cytotoxic agent is used alone or in combinationsof one or more cytotoxic agents such as calicheamycin, doxorubicin,taxol, methotrexate, gemcitabine, cytarabine, vinblastin, daunorubicin,docetaxel, irinotecan, epothilone B, epothilone D, cisplatin,carboplatin, and 5-fluoro-U.

[0064] The invention also provides methods of increasing thepermeability of a solid tumor to cytotoxic agents by administering atherapeutically effective amount of a PDGF-binding aptamer of theinvention. The invention also provides methods of increasing thepermeability of a solid tumor to cytotoxic agents by administering atherapeutically effective amount of a composition of the invention.

[0065] In one embodiment, the cancer or tumor is PDGF mediated cancer ortumor. In one embodiment, the PDGF mediated cancer or tumor is aglioblastoma, chronic myelomonocytic leukemia, a dermafibrosarcomaprotuberan, a gastrointestinal stromal tumor or a soft tissue sarcoma.

[0066] In one embodiment, the composition includes a cytotoxic agentbelonging to a class of cytotoxic agents selected from the groupconsisting of tubulin stabilizers, tubulin destabilizers,anti-metabolites, purine synthesis inhibitors, nucleoside analogs, DNAalkylating agents, DNA modifying agents, and vascular disrupting agents.In one embodiment, the cytotoxic agent is used alone or in combinationsof one or more cytotoxic agents such as calicheamycin, doxorubicin,taxol, methotrexate, gemcitabine, cytarabine, vinblastin, daunorubicin,docetaxel, irinotecan, epothilone B, epothilone D, cisplatin,carboplatin, and 5-fluoro-U.

[0067] The invention also provides methods of reducing constitutiveexpression of platelet derived growth factor in a tumor by administeringa therapeutically effective amount of a PDGF-binding aptamer of theinvention. The invention also provides methods of reducing constitutiveexpression of platelet derived growth factor in a tumor by administeringa therapeutically effective amount of a composition of the invention.

[0068] In one embodiment, the cancer or tumor is PDGF mediated cancer ortumor. In one embodiment, the PDGF mediated cancer or tumor is aglioblastoma, chronic myelomonocytic leukemia, a dermafibrosarcomaprotuberan, a gastrointestinal stromal tumor or a soft tissue sarcoma.

[0069] In one embodiment, the composition includes a cytotoxic agentbelonging to a class of cytotoxic agents selected from the groupconsisting of tubulin stabilizers, tubulin destabilizers,anti-metabolites, purine synthesis inhibitors, nucleoside analogs, DNAalkylating agents, DNA modifying agents, and vascular disrupting agents.In one embodiment, the cytotoxic agent is used alone or in combinationsof one or more cytotoxic agents such as calicheamycin, doxorubicin,taxol, methotrexate, gemcitabine, cytarabine, vinblastin, daunorubicin,docetaxel, irinotecan, epothilone B, epothilone D, cisplatin,carboplatin, and 5-fluoro-U.

[0070] The invention also provides methods of reducing angiogenesis andneovascularization in a tumor by administering a therapeuticallyeffective amount of a PDGF-binding aptamer of the invention. Theinvention also provides methods of reducing angiogenesis andneovascularization in a tumor by administering a therapeuticallyeffective amount of a composition of the invention.

[0071] In one embodiment, the cancer or tumor is PDGF mediated cancer ortumor. In one embodiment, the PDGF mediated cancer or tumor is aglioblastoma, chronic myelomonocytic leukemia, a dermafibrosarcomaprotuberan, a gastrointestinal stromal tumor or a soft tissue sarcoma.

[0072] In one embodiment, the composition includes a cytotoxic agentbelonging to a class of cytotoxic agents selected from the groupconsisting of tubulin stabilizers, tubulin destabilizers,anti-metabolites, purine synthesis inhibitors, nucleoside analogs, DNAalkylating agents, DNA modifying agents, and vascular disrupting agents.In one embodiment, the cytotoxic agent is used alone or in combinationsof one or more cytotoxic agents such as calicheamycin, doxorubicin,taxol, methotrexate, gemcitabine, cytarabine, vinblastin, daunorubicin,docetaxel, irinotecan, epothilone B, epothilone D, cisplatin,carboplatin, and 5-fluoro-U.

DETAILED DESCRIPTION OF THE INVENTION

[0073] The details of one or more embodiments of the invention are setforth in the accompanying description below. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. Other features, objects, and advantagesof the invention will be apparent from the description. In thespecification, the singular forms also include the plural unless thecontext clearly dictates otherwise. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. In the case of conflict, the present Specificationwill control.

[0074] The SELEX™ Method

[0075] A suitable method for generating an aptamer is with the processentitled “Systematic Evolution of Ligands by Exponential Enrichment”(“SELEX™”) generally depicted in FIG. 1. The SELEX™ process is a methodfor the in vitro evolution of nucleic acid molecules with highlyspecific binding to target molecules and is described in, e.g., U.S.patent application Ser. No. 07/536,428, filed Jun. 11, 1990, nowabandoned, U.S. Pat. No. 5,475,096 entitled “Nucleic Acid Ligands”, andU.S. Pat. No. 5,270,163 (see also WO 91/19813) entitled “Nucleic AcidLigands”. Each SELEX-identified nucleic acid ligand is a specific ligandof a given target compound or molecule. The SELEX™ process is based onthe unique insight that nucleic acids have sufficient capacity forforming a variety of two- and three-dimensional structures andsufficient chemical versatility available within their monomers to actas ligands (i.e., form specific binding pairs) with virtually anychemical compound, whether monomeric or polymeric. Molecules of any sizeor composition can serve as targets.

[0076] SELEX™ relies as a starting point upon a large library of singlestranded oligonucleotide templates comprising randomized sequencesderived from chemical synthesis on a standard DNA synthesizer. In someexamples, a population of 100% random oligonucleotides is screened. Inothers, each oligonucleotide in the population comprises a randomsequence and at least one fixed sequence at its 5′ and/or 3′ end whichcomprises a sequence shared by all the molecules of the oligonucleotidepopulation. Fixed sequences include sequences such as hybridizationsites for PCR primers, promoter sequences for RNA polymerases (e.g., T3,T4, T7, SP6, and the like), restriction sites, or homopolymericsequences, such as poly A or poly T tracts, catalytic cores, sites forselective binding to affinity columns, and other sequences to facilitatecloning and/or sequencing of an oligonucleotide of interest.

[0077] The random sequence portion of the oligonucleotide can be of anylength and can comprise ribonucleotides and/or deoxyribonucleotides andcan include modified or non-natural nucleotides or nucleotide analogs.See, e.g., U.S. Pat. Nos. 5,958,691; 5,660,985; 5,958,691; 5,698,687;5,817,635; and 5,672,695, PCT publication WO 92/07065. Randomoligonucleotides can be synthesized from phosphodiester-linkednucleotides using solid phase oligonucleotide synthesis techniques wellknown in the art (Froehler et al., Nucl. Acid Res. 14:5399-5467 (1986);Froehler et al., Tet. Lett. 27:5575-5578 (1986)). Oligonucleotides canalso be synthesized using solution phase methods such as triestersynthesis methods (Sood et al., Nucl. Acid Res. 4:2557 (1977); Hirose etal., Tet. Lett., 28:2449 (1978)). Typical syntheses carried out onautomated DNA synthesis equipment yield 10¹⁵-10¹⁷ molecules.Sufficiently large regions of random sequence in the sequence designincreases the likelihood that each synthesized molecule is likely torepresent a unique sequence.

[0078] To synthesize randomized sequences, mixtures of all fournucleotides are added at each nucleotide addition step during thesynthesis process, allowing for random incorporation of nucleotides. Inone embodiment, random oligonucleotides comprise entirely randomsequences; however, in other embodiments, random oligonucleotides cancomprise stretches of nonrandom or partially random sequences. Partiallyrandom sequences can be created by adding the four nucleotides indifferent molar ratios at each addition step.

[0079] Template molecules typically contain fixed 5′ and 3′ terminalsequences which flank an internal region of 30-50 random nucleotides. Astandard (1 μmole) scale synthesis will yield 10¹⁵-10¹⁶ individualtemplate molecules, sufficient for most SELEX experiments. The RNAlibrary is generated from this starting library by in vitrotranscription using recombinant T7 RNA polymerase. This library is thenmixed with the target under conditions favorable for binding andsubjected to step-wise iterations of binding, partitioning andamplification, using the same general selection scheme, to achievevirtually any desired criterion of binding affinity and selectivity.Starting from a mixture of nucleic acids, preferably comprising asegment of randomized sequence, the SELEX™ method includes steps ofcontacting the mixture with the target under conditions favorable forbinding, partitioning unbound nucleic acids from those nucleic acidswhich have bound specifically to target molecules, dissociating thenucleic acid-target complexes, amplifying the nucleic acids dissociatedfrom the nucleic acid-target complexes to yield a ligand-enrichedmixture of nucleic acids, then reiterating the steps of binding,partitioning, dissociating and amplifying through as many cycles asdesired to yield highly specific, high affinity nucleic acid ligands tothe target molecule.

[0080] Within a nucleic acid mixture containing a large number ofpossible sequences and structures, there is a wide range of bindingaffinities for a given target. A nucleic acid mixture comprising, forexample a 20 nucleotide randomized segment can have 4²⁰ candidatepossibilities. Those which have the higher affinity constants for thetarget are most likely to bind to the target. After partitioning,dissociation and amplification, a second nucleic acid mixture isgenerated, enriched for the higher binding affinity candidates.Additional rounds of selection progressively favor the best ligandsuntil the resulting nucleic acid mixture is predominantly composed ofonly one or a few sequences. These can then be cloned, sequenced andindividually tested for binding affinity as pure ligands.

[0081] Cycles of selection and amplification are repeated until adesired goal is achieved. In the most general case,selection/amplification is continued until no significant improvement inbinding strength is achieved on repetition of the cycle. The method maybe used to sample as many as about 10¹⁸ different nucleic acid species.The nucleic acids of the test mixture preferably include a randomizedsequence portion as well as conserved sequences necessary for efficientamplification. Nucleic acid sequence variants can be produced in anumber of ways including synthesis of randomized nucleic acid sequencesand size selection from randomly cleaved cellular nucleic acids. Thevariable sequence portion may contain fully or partially randomsequence; it may also contain sub portions of conserved sequenceincorporated with randomized sequence. Sequence variation in testnucleic acids can be introduced or increased by mutagenesis before orduring the selection/amplification iterations.

[0082] In one embodiment of SELEX™, the selection process is soefficient at isolating those nucleic acid ligands that bind moststrongly to the selected target, that only one cycle of selection andamplification is required. Such an efficient selection may occur, forexample, in a chromatographic-type process wherein the ability ofnucleic acids to associate with targets bound on a column operates insuch a manner that the column is sufficiently able to allow separationand isolation of the highest affinity nucleic acid ligands.

[0083] In many cases, it is not necessarily desirable to perform theiterative steps of SELEX™ until a single nucleic acid ligand isidentified. The target-specific nucleic acid ligand solution may includea family of nucleic acid structures or motifs that have a number ofconserved sequences and a number of sequences which can be substitutedor added without significantly affecting the affinity of the nucleicacid ligands to the target. By terminating the SELEX™ process prior tocompletion, it is possible to determine the sequence of a number ofmembers of the nucleic acid ligand solution family.

[0084] A variety of nucleic acid primary, secondary and tertiarystructures are known to exist. The structures or motifs that have beenshown most commonly to be involved in non-Watson-Crick type interactionsare referred to as hairpin loops, symmetric and asymmetric bulges,pseudoknots and myriad combinations of the same. Almost all known casesof such motifs suggest that they can be formed in a nucleic acidsequence of no more than 30 nucleotides. For this reason, it is oftenpreferred that SELEX procedures with contiguous randomized segments beinitiated with nucleic acid sequences containing a randomized segment ofbetween about 20-50 nucleotides.

[0085] The core SELEX™ method has been modified to achieve a number ofspecific objectives. For example, U.S. Pat. No. 5,707,796 describes theuse of SELEX™ in conjunction with gel electrophoresis to select nucleicacid molecules with specific structural characteristics, such as bentDNA. U.S. Pat. No. 5,763,177 describes SELEX™ based methods forselecting nucleic acid ligands containing photoreactive groups capableof binding and/or photocrosslinking to and/or photoinactivating a targetmolecule. U.S. Pat. No. 5,567,588 and U.S. application Ser. No.08/792,075, filed Jan. 31, 1997, entitled “Flow Cell SELEX”, describeSELEX™ based methods which achieve highly efficient partitioning betweenoligonucleotides having high and low affinity for a target molecule.U.S. Pat. No. 5,496,938 describes methods for obtaining improved nucleicacid ligands after the SELEX™ process has been performed. U.S. Pat. No.5,705,337 describes methods for covalently linking a ligand to itstarget.

[0086] SELEX™ can also be used to obtain nucleic acid ligands that bindto more than one site on the target molecule, and to obtain nucleic acidligands that include non-nucleic acid species that bind to specificsites on the target. SELEX™ provides means for isolating and identifyingnucleic acid ligands which bind to any envisionable target, includinglarge and small biomolecules including proteins (including both nucleicacid-binding proteins and proteins not known to bind nucleic acids aspart of their biological function) cofactors and other small molecules.For example, see U.S. Pat. No. 5,580,737 which discloses nucleic acidsequences identified through SELEX™ which are capable of binding withhigh affinity to caffeine and the closely related analog, theophylline.

[0087] Counter-SELEX™ is a method for improving the specificity ofnucleic acid ligands to a target molecule by eliminating nucleic acidligand sequences with cross-reactivity to one or more non-targetmolecules. Counter-SELEX™ is comprised of the steps of a) preparing acandidate mixture of nucleic acids; b) contacting the candidate mixturewith the target, wherein nucleic acids having an increased affinity tothe target relative to the candidate mixture may be partitioned from theremainder of the candidate mixture; c) partitioning the increasedaffinity nucleic acids from the remainder of the candidate mixture; d)contacting the increased affinity nucleic acids with one or morenon-target molecules such that nucleic acid ligands with specificaffinity for the non-target molecule(s) are removed; and e) amplifyingthe nucleic acids with specific affinity to the target molecule to yielda mixture of nucleic acids enriched for nucleic acid sequences with arelatively higher affinity and specificity for binding to the targetmolecule.

[0088] One potential problem encountered in the use of nucleic acids astherapeutics and vaccines is that oligonucleotides in theirphosphodiester form may be quickly degraded in body fluids byintracellular and extracellular enzymes such as endonucleases andexonucleases before the desired effect is manifest. The SELEX methodthus encompasses the identification of high-affinity nucleic acidligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX-identified nucleic acid ligands containingmodified nucleotides are described in U.S. Pat. No. 5,660,985, whichdescribes oligonucleotides containing nucleotide derivatives chemicallymodified at the 2′ position of ribose, 5 position of pyrimidines, and 8position of purines. U.S. Pat. No. 5,756,703 describes oligonucleotidescontaining various 2′-modified pyrimidines. U.S. Pat. No. 5,580,737describes highly specific nucleic acid ligands containing one or morenucleotides modified with 2′-amino (2′-NH2), 2′-fluoro (2′-F), and/or2′-O-methyl (2′-OMe) substituents.

[0089] Modifications of the nucleic acid ligands contemplated in thisinvention include, but are not limited to, those which provide otherchemical groups that incorporate additional charge, polarizability,hydrophobicity, hydrogen bonding, electrostatic interaction, andfluxionality to the nucleic acid ligand bases or to the nucleic acidligand as a whole. Such modifications include, but are not limited to,2′-position sugar modifications, 5-position pyrimidine modifications,8-position purine modifications, modifications at exocyclic amines,substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil;backbone modifications, phosphorothioate or alkyl phosphatemodifications, methylations, unusual base-pairing combinations such asthe isobases isocytidine and isoguanidine and the like. Modificationscan also include 3′ and 5′ modifications such as capping.

[0090] The modifications can be pre- or post-SELEX processmodifications. Pre-SELEX process modifications yield nucleic acidligands with both specificity for their SELEX target and improved invivo stability. Post-SELEX process modifications made to 2′-OH nucleicacid ligands can result in improved in vivo stability without adverselyaffecting the binding capacity of the nucleic acid ligand.

[0091] Other modifications are known to one of ordinary skill in theart. Such modifications may be made post-SELEX process (modification ofpreviously identified unmodified ligands) or by incorporation into theSELEX process.

[0092] The SELEX method encompasses combining selected oligonucleotideswith other selected oligonucleotides and non-oligonucleotide functionalunits as described in U.S. Pat. No. 5,637,459 and U.S. Pat. No.5,683,867. The SELEX method further encompasses combining selectednucleic acid ligands with lipophilic or non-immunogenic high molecularweight compounds in a diagnostic or therapeutic complex, as described inU.S. Pat. No. 6,011,020. VEGF nucleic acid ligands that are associatedwith a lipophilic compound, such as diacyl glycerol or dialkyl glycerol,in a diagnostic or therapeutic complex are described in U.S. Pat. No.5,859,228.

[0093] VEGF nucleic acid ligands that are associated with a lipophiliccompound, such as a glycerol lipid, or a non-immunogenic high molecularweight compound, such as polyalkylene glycol are further described inU.S. Pat. No. 6,051,698. VEGF nucleic acid ligands that are associatedwith a non-immunogenic, high molecular weight compound or a lipophiliccompound are further described in PCT Publication No. WO 98/18480. Thesepatents and applications allow the combination of a broad array ofshapes and other properties, and the efficient amplification andreplication properties, of oligonucleotides with the desirableproperties of other molecules.

[0094] The identification of nucleic acid ligands to small, flexiblepeptides via the SELEX method has also been explored. Small peptideshave flexible structures and usually exist in solution in an equilibriumof multiple conformers, and thus it was initially thought that bindingaffinities may be limited by the conformational entropy lost uponbinding a flexible peptide. However, the feasibility of identifyingnucleic acid ligands to small peptides in solution was demonstrated inU.S. Pat. No. 5,648,214. In this patent, high affinity RNA nucleic acidligands to substance P, an 11 amino acid peptide, were identified.

[0095] To generate oligonucleotide populations which are resistant tonucleases and hydrolysis, modified oligonucleotides can be used and caninclude one or more substitute internucleotide linkages, altered sugars,altered bases, or combinations thereof. In one embodiment,oligonucleotides are provided in which the P(O)O group is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), P(O)NR₂ (“amidate”), P(O)R,P(O)OR′, CO or CH₂ (“formacetal”) or 3′-amine (—NH—CH₂—CH₂—), whereineach R or R′ is independently H or substituted or unsubstituted alkyl.Linkage groups can be attached to adjacent nucleotide through an —O—,—N—, or —S— linkage. Not all linkages in the oligonucleotide arerequired to be identical.

[0096] In further embodiments, the oligonucleotides comprise modifiedsugar groups, for example, one or more of the hydroxyl groups isreplaced with halogen, aliphatic groups, or functionalized as ethers oramines. In one embodiment, the 2′-position of the furanose residue issubstituted by any of an O-methyl, O-alkyl, O-allyl, S-alkyl, S-allyl,or halo group. Methods of synthesis of 2′-modified sugars are describedin Sproat, et al., Nucl. Acid Res. 19:733-738 (1991); Cotten, et al.,Nucl. Acid Res. 19:2629-2635 (1991); and Hobbs, et al., Biochemistry12:5138-5145 (1973). The use of 2-fluoro-ribonucleotide oligomermolecules can increase the sensitivity of a nucleic acid molecule for atarget molecule by ten- to- one hundred-fold over those generated usingunsubstituted ribo- or deoxyribooligonucleotides (Pagratis, et al., Nat.Biotechnol. 15:68-73 (1997)), providing additional binding interactionswith a target molecule and increasing the stability of the secondarystructure(s) of the nucleic acid molecule (Kraus, et al., Journal ofImmunology 160:5209-5212 (1998); Pieken, et al., Science 253:314-317(1991); Lin, et al., Nucl. Acids Res. 22:5529-5234 (1994); Jellinek, etal. Biochemistry 34:11363-11372 (1995); Pagratis, et al., Nat.Biotechnol 15:68-73 (1997)).

[0097] Nucleic acid aptamer molecules are generally selected in a 5 to20 cycle procedure. In one embodiment, heterogeneity is introduced onlyin the initial selection stages and does not occur throughout thereplicating process.

[0098] The starting library of DNA sequences is generated by automatedchemical synthesis on a DNA synthesizer. This library of sequences istranscribed in vitro into RNA using T7 RNA polymerase or modified T7 RNApolymerases and purified. In one example, the 5′-fixed:random:3′-fixedsequence is separated by random sequence having 30 to 50 nucleotides.

[0099] The aptamers with specificity and binding affinity to the targetsof the present invention are selected by the SELEX process describedabove. As part of the SELEX process the sequences selected to bind tothe target are then optionally minimized to determine the minimalsequence having binding affinity, and optimized by performing random ordirected mutagenesis of the minimized sequence to determine if increasesof affinity or alternatively which positions in the sequence areessential for binding activity. Additionally, selections can beperformed with sequences incorporating modified sequences to stabilizethe aptamer molecules against degradation in vivo.

2′Modified SELEX™

[0100] In addition, the SELEX™ method can be performed to generate2′modified aptamers as described in U.S. Ser. No. 60/430,761, filed Dec.3, 2002, U.S. Provisional Patent Application Ser. No. 60/487,474, filedJul. 15, 2003, and U.S. Provisional Patent Application Ser. No.60/517,039, filed Nov. 4, 2003, and U.S. patent application Ser. No.10/29,581, filed Dec. 3, 2003, each of which is herein incorporated byreference in its entirety.

[0101] In order for an aptamer to be suitable for use as a therapeutic,it is preferably inexpensive to synthesize, safe and stable in vivo.Wild-type RNA and DNA aptamers are typically not stable in vivo becauseof their susceptibility to degradation by nucleases. Resistance tonuclease degradation can be greatly increased by the incorporation ofmodifying groups at the 2′-position. Fluoro and amino groups have beensuccessfully incorporated into oligonucleotide libraries from whichaptamers have been subsequently selected. However, these modificationsgreatly increase the cost of synthesis of the resultant aptamer, and mayintroduce safety concerns because of the possibility that the modifiednucleotides could be recycled into host DNA, by degradation of themodified oligonucleotides and subsequent use of the nucleotides assubstrates for DNA synthesis.

[0102] Aptamers that contain 2′-O-methyl (2′-OMe) nucleotides overcomemany of these drawbacks. Oligonucleotides containing 2′-O-methylnucleotides are nuclease-resistant and inexpensive to synthesize.Although 2′-O-methyl nucleotides are ubiquitous in biological systems,natural polymerases do not accept 2′-O-methyl NTPs as substrates underphysiological conditions, thus there are no safety concerns over therecycling of 2′-O-methyl nucleotides into host DNA.

[0103] The present invention also provides materials and methods toproduce stabilized oligonucleotides, including, e.g., aptamers, whichcontain modified nucleotides (e.g., nucleotides which have amodification at the 2′position) which make the oligonucleotide morestable than the unmodified oligonucleotide. The stabilizedoligonucleotides produced by the materials and methods of the presentinvention are also more stable to enzymatic and chemical degradation aswell as thermal and physical degradation. For example, oligonucleotidescontaining 2′-O-methyl nucleotides are nuclease-resistant andinexpensive to synthesize. Although 2′-O-methyl nucleotides areubiquitous in biological systems, natural polymerases do not accept2′-O-methyl NTPs as substrates under physiological conditions, thusthere are no safety concerns over the recycling of 2′-O-methylnucleotides into host DNA.

[0104] In one embodiment, the present invention provides combinations of2′-OH, 2′-F, 2′-deoxy, and 2′-OMe modifications of the ATP, GTP, CTP,TTP, and UTP nucleotides. In another embodiment, the present inventionprovides combinations of 2′-OH, 2′-F, 2′-deoxy, 2′-OMe, 2′-NH₂, and2′-methoxyethyl modifications of the ATP, GTP, CTP, TTP, and UTPnucleotides. In one embodiment, the present invention provides 56combinations of 2′-OH, 2′-F, 2′-deoxy, 2′-OMe, 2′-NH₂, and2′-methoxyethyl modifications the ATP, GTP, CTP, TTP, and UTPnucleotides.

[0105] 2′ modified aptamers of the invention are created using modifiedpolymerases, such as, e.g., a modified T7 polymerase, having a higherincorporation rate of modified nucleotides having bulky substituents atthe furanose 2′ position, than wild-type polymerases. For example, adouble T7 polymerase mutant (Y639F/H784A) having the histidine atposition 784 changed to an alanine, or other small amino acid, residue,in addition to the Y639F mutation has been described for incorporationof bulky 2′ substituents and has been used to incorporate modifiedpyrimidine NTPs. A single mutant T7 polymerase (H784A) having thehistidine at position 784 changed to an alanine residue has also beendescribed. (Padilla et al., Nucleic Acids Research, 2002, 30: 138). Inboth the Y639F/H784A double mutant and H784A single mutant T7polymerases, the change to smaller amino acid residues allows for theincorporation of bulkier nucleotide substrates, e.g., 2′-O methylsubstituted nucleotides.

[0106] Another important factor in the production of 2′-modifiedaptamers is the use of both divalent magnesium and manganese in thetranscription mixture. Different combinations of concentrations ofmagnesium chloride and manganese chloride have been found to affectyields of 2′-O-methylated transcripts, the optimum concentration of themagnesium and manganese chloride being dependent on the concentration inthe transcription reaction mixture of NTPs which complex divalent metalions.

[0107] Priming transcription with GMP or guanosine is also important.This effect results from the specificity of the polymerase for theinitiating nucleotide. As a result, the 5′-terminal nucleotide of anytranscript generated in this fashion is likely to be 2′-OH G. Thepreferred concentration of GMP (or guanosine) is 0.5 mM and even morepreferably 1 mM. It has also been found that including PEG, preferablyPEG-8000, in the transcription reaction is useful to maximizeincorporation of modified nucleotides.

[0108] There are several examples of 2′-OMe containing aptamers in theliterature, see, for example Green et al., Current Biology 2, 683-695,1995. These were generated by the in vitro selection of libraries ofmodified transcripts in which the C and U residues were 2′-fluoro (2′-F)substituted and the A and G residues were 2′-OH. Once functionalsequences were identified then each A and G residue was tested fortolerance to 2′-OMe substitution, and the aptamer was re-synthesizedhaving all A and G residues which tolerated 2′-OMe substitution as2′-OMe residues. Most of the A and G residues of aptamers generated inthis two-step fashion tolerate substitution with 2′-OMe residues,although, on average, approximately 20% do not. Consequently, aptamersgenerated using this method tend to contain from two to four 2′-OHresidues, and stability and cost of synthesis are compromised as aresult. By incorporating modified nucleotides into the transcriptionreaction which generate stabilized oligonucleotides used inoligonucleotide libraries from which aptamers are selected and enrichedby SELEX™ (and/or any of its variations and improvements, includingthose described below), the methods of the present invention eliminatethe need for stabilizing the selected aptamer oligonucleotides (e.g., byresynthesizing the aptamer oligonucleotides with modified nucleotides).

[0109] Furthermore, the modified oligonucleotides of the invention canbe further stabilized after the selection process has been completed.(See “post-SELEX™ modifications”, including truncating, deleting andmodification, below.)

[0110] To generate oligonucleotide populations which are resistant tonucleases and hydrolysis, modified oligonucleotides can be used and caninclude one or more substitute internucleotide linkages, altered sugars,altered bases, or combinations thereof. In one embodiment,oligonucleotides are provided in which the P(O)O group is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), P(O)NR₂ (“amidate”), P(O)R,P(O)OR′, CO or CH₂ (“formacetal”) or 3′-amine (—NH—CH₂—CH₂—), whereineach R or R′ is independently H or substituted or unsubstituted alkyl.Linkage groups can be attached to adjacent nucleotide through an —O—,—N—, or —S— linkage. Not all linkages in the oligonucleotide arerequired to be identical.

[0111] Incorporation of modified nucleotides into the aptamers of theinvention is accomplished before (pre-) the selection process (e.g., apre-SELEX™ process modification). Optionally, aptamers of the inventionin which modified nucleotides have been incorporated by pre-SELEX™process modification can be further modified by post-SELEX™ processmodification (i.e., a post-SELEX™ process modification after apre-SELEX™ modification). Pre-SELEX™ process modifications yieldmodified nucleic acid ligands with specificity for the SELEX™ target andalso improved in vivo stability. Post-SELEX™ process modifications(e.g., modification of previously identified ligands having nucleotidesincorporated by pre-SELEX™ process modification) can result in a furtherimprovement of in vivo stability without adversely affecting the bindingcapacity of the nucleic acid ligand having nucleotides incorporated bypre-SELEX™ process modification.

[0112] Modified Polymerases

[0113] A single mutant T7 polymerase (Y639F) in which the tyrosineresidue at position 639 has been changed to phenylalanine readilyutilizes 2′deoxy, 2′amino-, and 2′fluoro-nucleotide triphosphates (NTPs)as substrates and has been widely used to synthesize modified RNAs for avariety of applications. However, this mutant T7 polymerase reportedlycan not readily utilize (e.g., incorporate) NTPs with bulkier2′-substituents, such as 2′-O-methyl (2′-OMe) or 2′-azido (2′-N₃)substituents. For incorporation of bulky 2′ substituents, a double T7polymerase mutant (Y639F/H784A) having the histidine at position 784changed to an alanine, or other small amino acid, residue, in additionto the Y639F mutation has been described and has been used toincorporate modified pyrimidine NTPs. A single mutant T7 polymerase(H784A) having the histidine at position 784 changed to an alanineresidue has also been described. (Padilla et al., Nucleic AcidsResearch, 2002, 30: 138). In both the Y639F/H784A double mutant andH784A single mutant T7 polymerases, the change to smaller amino acidresidues allows for the incorporation of bulkier nucleotide substrates,e.g., 2′-O methyl substituted nucleotides.

[0114] The present invention provides methods and conditions for usingthese and other modified T7 polymerases having a higher incorporationrate of modified nucleotides having bulky substituents at the furanose2′ position, than wild-type polymerases. Generally, it has been foundthat under the conditions disclosed herein, the Y693F single mutant canbe used for the incorporation of all 2′-OMe substituted NTPs except GTPand the Y639F/H784A double mutant can be used for the incorporation ofall 2′-OMe substituted NTPs including GTP. It is expected that the H784Asingle mutant possesses similar properties when used under theconditions disclosed herein.

[0115] The present invention provides methods and conditions formodified T7 polymerases to enzymatically incorporate modifiednucleotides into oligonucleotides. Such oligonucleotides may besynthesized entirely of modified nucleotides, or with a subset ofmodified nucleotides. The modifications can be the same or different.All nucleotides may be modified, and all may contain the samemodification. All nucleotides may be modified, but contain differentmodifications, e.g., all nucleotides containing the same base may haveone type of modification, while nucleotides containing other bases mayhave different types of modification. All purine nucleotides may haveone type of modification (or are unmodified), while all pyrimidinenucleotides have another, different type of modification (or areunmodified). In this way, transcripts, or libraries of transcripts aregenerated using any combination of modifications, for example,ribonucleotides, (2′-OH, “rN”), deoxyribonucleotides (2′-deoxy), 2′-F,and 2′-OMe nucleotides. A mixture containing 2′-OMe C and U and 2′-OH Aand G is called “rRmY”; a mixture containing deoxy A and G and 2′-OMe Uand C is called “dRmY”; a mixture containing 2′-OMe A, C, and U, and2′-OH G is called “rGmH”; a mixture alternately containing 2′-OMe A, C,U and G and 2′-OMe A, U and C and 2′-F G is called “toggle”; a mixturecontaining 2′-OMe A, U, C, and G, where up to 10% of the G's are deoxyis called “r/mGmH”; a mixture containing 2′-O Me A, U, and C, and 2′-F Gis called “fGmH”; and a mixture containing deoxy A, and 2′-OMe C, G andU is called “dAmB”.

[0116] A preferred embodiment includes any combination of 2′-OH,2′-deoxy and 2′-OMe nucleotides. A more preferred embodiment includesany combination of 2′-deoxy and 2′-OMe nucleotides. An even morepreferred embodiment is with any combination of 2′-deoxy and 2′-OMenucleotides in which the pyrimidines are 2′-OMe (such as dRmY, mN ordGmH).

2′-O-Methyl Modified Nucleotide SELEX™

[0117] The present invention provides methods to generate libraries of2′-modified (e.g., 2′-OMe) RNA transcripts in conditions under which apolymerase accepts 2′-modified NTPs. Preferably, the polymerase is theY693F/H784A double mutant or the Y693F single mutant. Other polymerases,particularly those that exhibit a high tolerance for bulky2′-substituents, may also be used in the present invention. Suchpolymerases can be screened for this capability by assaying theirability to incorporate modified nucleotides under the transcriptionconditions disclosed herein. A number of factors have been determined tobe crucial for the transcription conditions useful in the methodsdisclosed herein. For example, great increases in the yields of modifiedtranscript are observed when a leader sequence is incorporated into the5′ end of a fixed sequence at the 5′ end of the DNA transcriptiontemplate, such that at least about the first 6 residues of the resultanttranscript are all purines.

[0118] Another important factor in obtaining transcripts incorporatingmodified nucleotides is the presence or concentration of 2′-OH GTP.Transcription can be divided into two phases: the first phase isinitiation, during which an NTP is added to the 3′-hydroxyl end of GTP(or another substituted guanosine) to yield a dinucleotide which is thenextended by about 10-12 nucleotides, the second phase is elongation,during which transcription proceeds beyond the addition of the firstabout 10-12 nucleotides. It has been found that small amounts of 2′-OHGTP added to a transcription mixture containing an excess of 2′-OMe GTPare sufficient to enable the polymerase to initiate transcription using2′-OH GTP, but once transcription enters the elongation phase thereduced discrimination between 2′-OMe and 2′-OH GTP, and the excess of2′-OMe GTP over 2′-OH GTP allows the incorporation of principally the2′-OMe GTP.

[0119] Another important factor in the incorporation of 2′-OMe intotranscripts is the use of both divalent magnesium and manganese in thetranscription mixture. Different combinations of concentrations ofmagnesium chloride and manganese chloride have been found to affectyields of 2′-O-methylated transcripts, the optimum concentration of themagnesium and manganese chloride being dependent on the concentration inthe transcription reaction mixture of NTPs which complex divalent metalions. To obtain the greatest yields of maximally 2′ substitutedO-methylated transcripts (i.e., all A, C, and U and about 90% of Gnucleotides), concentrations of approximately 5 mM magnesium chlorideand 1.5 mM manganese chloride are preferred when each NTP is present ata concentration of 0.5 mM. When the concentration of each NTP is 1.0 mM,concentrations of approximately 6.5 mM magnesium chloride and 2.0 mMmanganese chloride are preferred. When the concentration of each NTP is2.0 mM, concentrations of approximately 9.6 mM magnesium chloride and2.9 mM manganese chloride are preferred. In any case, departures fromthese concentrations of up to two-fold still give significant amounts ofmodified transcripts.

[0120] Priming transcription with GMP or guanosine is also important.This effect results from the specificity of the polymerase for theinitiating nucleotide. As a result, the 5′-terminal nucleotide of anytranscript generated in this fashion is likely to be 2′-OH G. Thepreferred concentration of GMP (or guanosine) is 0.5 mM and even morepreferably 1 mM. It has also been found that including PEG, preferablyPEG-8000, in the transcription reaction is useful to maximizeincorporation of modified nucleotides.

[0121] For maximum incorporation of 2′-OMe ATP (100%), UTP(100%),CTP(100%) and GTP (˜90%) (“r/mGmH”) into transcripts the followingconditions are preferred: HEPES buffer 200 mM, DTT 40 mM, spermidine 2mM, PEG-8000 10% (w/v), Triton X-100 0.01% (w/v), MgCl₂ 5 mM (6.5 mMwhere the concentration of each 2′-OMe NTP is 1.0 mM), MnCl₂ 1.5 mM (2.0mM where the concentration of each 2′-OMe NTP is 1.0 mM), 2′-OMe NTP(each) 500 μM (more preferably, 1.0 mM), 2′-OH GTP 30 μM, 2′-OH GMP 500μM, pH 7.5, Y639F/H784A T7 RNA Polymerase 15 units/ml, inorganicpyrophosphatase 5 units/ml, and an all-purine leader sequence of atleast 8 nucleotides long. As used herein, one unit of the Y639F/H784Amutant T7 RNA polymerase, or any other mutant T7 RNA polymerasespecified herein) is defined as the amount of enzyme required toincorporate 1 nmole of 2′-OMe NTPs into transcripts under the r/mGmHconditions. As used herein, one unit of inorganic pyrophosphatase isdefined as the amount of enzyme that will liberate 1.0 mole of inorganicorthophosphate per minute at pH 7.2 and 25° C.

[0122] For maximum incorporation (100%) of 2′-OMe ATP, UTP and CTP(“rGmH”) into transcripts the following conditions are preferred: HEPESbuffer 200 mM, DTT 40 mM, spermidine 2 mM, PEG-8000 10% (w/v), TritonX-100 0.01% (w/v), MgCl₂ 5 mM (9.6 mM where the concentration of each2′-OMe NTP is 2.0 mM), MnCl₂ 1.5 mM (2.9 mM where the concentration ofeach 2′-OMe NTP is 2.0 mM), 2′-OMe NTP (each) 500 μM (more preferably,2.0 mM), pH 7.5, Y639F T7 RNA Polymerase 15 units/ml, inorganicpyrophosphatase 5 units/ml, and an all-purine leader sequence of atleast 8 nucleotides long.

[0123] For maximum incorporation (100%) of 2′-OMe UTP and CTP (“rRmY”)into transcripts the following conditions are preferred: HEPES buffer200 mM, DTT 40 mM, spermidine 2 mM, PEG-8000 10% (w/v), Triton X-1000.01% (w/v), MgCl₂ 5 mM (9.6 mM where the concentration of each 2′-OMeNTP is 2.0 mM), MnCl₂ 1.5 mM (2.9 mM where the concentration of each2′-OMe NTP is 2.0 mM), 2′-OMe NTP (each) 500 μM (more preferably, 2.0mM), pH 7.5, Y639F/H784A T7 RNA Polymerase 15 units/ml, inorganicpyrophosphatase 5 units/ml, and an all-purine leader sequence of atleast 8 nucleotides long.

[0124] For maximum incorporation (100%) of deoxy ATP and GTP and 2′-OMeUTP and CTP (“dRmY”) into transcripts the following conditions arepreferred: HEPES buffer 200 mM, DTT 40 mM, spermine or spermidine 2 mM,PEG-8000 10% (w/v), Triton X-100 0.01% (w/v), MgCl₂ 9.6 mM, MnCl₂ 2.9mM, 2′-OMe NTP (each) 2.0 mM, pH 7.5, Y639F T7 RNA Polymerase 15units/ml, inorganic pyrophosphatase 5 units/ml, and an all-purine leadersequence of at least 8 nucleotides long.

[0125] For maximum incorporation (100%) of 2′-OMe ATP, UTP and CTP and2′-F GTP (“fGmH”) into transcripts the following conditions arepreferred: HEPES buffer 200 mM, DTT 40 mM, spermidine 2 mM, PEG-8000 10%(w/v), Triton X-100 0.01% (w/v), MgCl₂ 9.6 mM, MnCl₂ 2.9 mM, 2′-OMe NTP(each) 2.0 mM, pH 7.5, Y639F T7 RNA Polymerase 15 units/ml, inorganicpyrophosphatase 5 units/ml, and an all-purine leader sequence of atleast 8 nucleotides long.

[0126] For maximum incorporation (100%) of deoxy ATP and 2′-OMe UTP, GTPand CTP (“dAmB”) into transcripts the following conditions arepreferred: HEPES buffer 200 mM, DTT 40 mM, spermidine 2 mM, PEG-8000 10%(w/v), Triton X-100 0.01% (w/v), MgCl₂ 9.6 mM, MnCl₂ 2.9 mM, 2′-OMe NTP(each) 2.0 mM, pH 7.5, Y639F T7 RNA Polymerase 15 units/ml, inorganicpyrophosphatase 5 units/ml, and an all-purine leader sequence of atleast 8 nucleotides long.

[0127] For each of the above, (1) transcription is preferably performedat a temperature of from about 30° C. to about 45° C. and for a periodof at least two hours and (2) 50-300 nM of a double stranded DNAtranscription template is used (200 nm template was used for round 1 toincrease diversity (300 nm template was used for dRmY transcriptions),and for subsequent rounds approximately 50 nM, a {fraction (1/10)}dilution of an optimized PCR reaction, using conditions describedherein, was used). The preferred DNA transcription templates aredescribed below (where ARC254 and ARC256 transcribe under all 2′-OMeconditions and ARC255 transcribes under rRmY conditions). ARC254:5′-CATCGATGCTAGTCGTAACGATCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCGAGAACGTTCTCTCCTCTCCCTATAGTGAGTCGTATTA-3′ (SEQ ID NO: 51) ARC255:5′-CATGCATCGCGACTGACTAGCCGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGTAGAACGTTCTCTCCTCTCCCTATAGTGAGTCGTATTA-3′ (SEQ ID NO: 52) ARC256:5′-CATCGATCGATCGATCGACAGCGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGTAGAACGTTCTCTCCTCTCCCTATAGTGAGTCGTATTA-3′ (SEQ ID NO: 53)

[0128] Under rN transcription conditions of the present invention, thetranscription reaction mixture comprises 2′-OH adenosine triphosphates(ATP), 2′-OH guanosine triphosphates (GTP), 2′-OH cytidine triphosphates(CTP), and 2′-OH uridine triphosphates (UTP). The modifiedoligonucleotides produced using the rN transcription mixtures of thepresent invention comprise substantially all 2′-OH adenosine, 2′-OHguanosine, 2′-OH cytidine, and 2′-OH uridine. In a preferred embodimentof rN transcription, the resulting modified oligonucleotides comprise asequence where at least 80% of all adenosine nucleotides are 2′-OHadenosine, at least 80% of all guanosine nucleotides are 2′-OHguanosine, at least 80% of all cytidine nucleotides are 2′-OH cytidine,and at least 80% of all uridine nucleotides are 2′-OH uridine. In a morepreferred embodiment of rN transcription, the resulting modifiedoligonucleotides of the present invention comprise a sequence where atleast 90% of all adenosine nucleotides are 2′-OH adenosine, at least 90%of all guanosine nucleotides are 2′-OH guanosine, at least 90% of allcytidine nucleotides are 2′-OH cytidine, and at least 90% of all uridinenucleotides are 2′-OH uridine. In a most preferred embodiment of rNtranscription, the modified oligonucleotides of the present inventioncomprise 100% of all adenosine nucleotides are 2′-OH adenosine, of allguanosine nucleotides are 2′-OH guanosine, of all cytidine nucleotidesare 2′-OH cytidine, and of all uridine nucleotides are 2′-OH uridine.

[0129] Under rRmY transcription conditions of the present invention, thetranscription reaction mixture comprises 2′-OH adenosine triphosphates,2′-OH guanosine triphosphates, 2′-O-methyl cytidine triphosphates, and2′-O-methyl uridine triphosphates. The modified oligonucleotidesproduced using the rRmY transcription mixtures of the present inventioncomprise substantially all 2′-OH adenosine, 2′-OH guanosine, 2′-O-methylcytidine and 2′-O-methyl uridine. In a preferred embodiment, theresulting modified oligonucleotides comprise a sequence where at least80% of all adenosine nucleotides are 2′-OH adenosine, at least 80% ofall guanosine nucleotides are 2′-OH guanosine, at least 80% of allcytidine nucleotides are 2′-O-methyl cytidine and at least 80% of alluridine nucleotides are 2′-O-methyl uridine. In a more preferredembodiment, the resulting modified oligonucleotides comprise a sequencewhere at least 90% of all adenosine nucleotides are 2′-OH adenosine, atleast 90% of all guanosine nucleotides are 2′-OH guanosine, at least 90%of all cytidine nucleotides are 2′-O-methyl cytidine and at least 90% ofall uridine nucleotides are 2′-O-methyl uridine In a most preferredembodiment, the resulting modified oligonucleotides comprise a sequencewhere 100% of all adenosine nucleotides are 2′-OH adenosine, 100% of allguanosine nucleotides are 2′-OH guanosine, 100% of all cytidinenucleotides are 2′-O-methyl cytidine and 100% of all uridine nucleotidesare 2′-O-methyl uridine.

[0130] Under dRmY transcription conditions of the present invention, thetranscription reaction mixture comprises 2′-deoxy purine triphosphatesand 2′-O-methyl pyrimidine triphosphates. The modified oligonucleotidesproduced using the dRmY transcription conditions of the presentinvention comprise substantially all 2′-deoxy purines and 2′-O-methylpyrimidines. In a preferred embodiment, the resulting modifiedoligonucleotides of the present invention comprise a sequence where atleast 80% of all purine nucleotides are 2′-deoxy purines and at least80% of all pyrimidine nucleotides are 2′-O-methyl pyrimidines. In a morepreferred embodiment, the resulting modified oligonucleotides of thepresent invention comprise a sequence where at least 90% of all purinenucleotides are 2′-deoxy purines and at least 90% of all pyrimidinenucleotides are 2′-O-methyl pyrimidines. In a most preferred embodiment,the resulting modified oligonucleotides of the present inventioncomprise a sequence where 100% of all purine nucleotides are 2′-deoxypurines and 100% of all pyrimidine nucleotides are 2′-O-methylpyrimidines.

[0131] Under rGmH transcription conditions of the present invention, thetranscription reaction mixture comprises 2′-OH guanosine triphosphates,2′-O-methyl cytidine triphosphates, 2′-O-methyl uridine triphosphates,and 2′-O-methyl adenosine triphosphates. The modified oligonucleotidesproduced using the rGmH transcription mixtures of the present inventioncomprise substantially all 2′-OH guanosine, 2′-O-methyl cytidine,2′-O-methyl uridine, and 2′-O-methyl adenosine. In a preferredembodiment, the resulting modified oligonucleotides comprise a sequencewhere at least 80% of all guanosine nucleotides are 2′-OH guanosine, atleast 80% of all cytidine nucleotides are 2′-O-methyl cytidine, at least80% of all uridine nucleotides are 2′-O-methyl uridine, and at least 80%of all adenosine nucleotides are 2′-O-methyl adenosine. In a morepreferred embodiment, the resulting modified oligonucleotides comprise asequence where at least 90% of all guanosine nucleotides are 2′-OHguanosine, at least 90% of all cytidine nucleotides are 2′-O-methylcytidine, at least 90% of all uridine nucleotides are 2′-O-methyluridine, and at least 90% of all adenosine nucleotides are 2′-O-methyladenosine. In a most preferred embodiment, the resulting modifiedoligonucleotides comprise a sequence where 100% of all guanosinenucleotides are 2′-OH guanosine, 100% of all cytidine nucleotides are2′-O-methyl cytidine, 100% of all uridine nucleotides are 2′-O-methyluridine, and 100% of all adenosine nucleotides are 2′-O-methyladenosine.

[0132] Under r/mGmH transcription conditions of the present invention,the transcription reaction mixture comprises 2′-O-methyl adenosinetriphosphate, 2′-O-methyl cytidine triphosphate, 2′-O-methyl guanosinetriphosphate, 2′-O-methyl uridine triphosphate and deoxy guanosinetriphosphate. The resulting modified oligonucleotides produced using ther/mGmH transcription mixtures of the present invention comprisesubstantially all 2′-O-methyl adenosine, 2′-O-methyl cytidine,2′-O-methyl guanosine, and 2′-O-methyl uridine, wherein the populationof guanosine nucleotides has a maximum of about 10% deoxy guanosine. Ina preferred embodiment, the resulting r/mGmH modified oligonucleotidesof the present invention comprise a sequence where at least 80% of alladenosine nucleotides are 2′-O-methyl adenosine, at least 80% of allcytidine nucleotides are 2′-O-methyl cytidine, at least 80% of allguanosine nucleotides are 2′-O-methyl guanosine, at least 80% of alluridine nucleotides are 2′-O-methyl uridine, and no more than about 10%of all guanosine nucleotides are deoxy guanosine. In a more preferredembodiment, the resulting modified oligonucleotides comprise a sequencewhere at least 90% of all adenosine nucleotides are 2′-O-methyladenosine, at least 90% of all cytidine nucleotides are 2′-O-methylcytidine, at least 90% of all guanosine nucleotides are 2′-O-methylguanosine, at least 90% of all uridine nucleotides are 2′-O-methyluridine, and no more than about 10% of all guanosine nucleotides aredeoxy guanosine. In a most preferred embodiment, the resulting modifiedoligonucleotides comprise a sequence where 100% of all adenosinenucleotides are 2′-O-methyl adenosine, 100% of all cytidine nucleotidesare 2′-O-methyl cytidine, 90% of all guanosine nucleotides are2′-O-methyl guanosine, and 100% of all uridine nucleotides are2′-O-methyl uridine, and no more than about 10% of all guanosinenucleotides are deoxy guanosine.

[0133] Under fGmH transcription conditions of the present invention, thetranscription reaction mixture comprises 2′-O-methyl adenosinetriphosphates (ATP), 2′-O-methyl uridine triphosphates (UTP),2′-O-methyl cytidine triphosphates (CTP), and 2′-F guanosinetriphosphates. The modified oligonucleotides produced using the fGmHtranscription conditions of the present invention comprise substantiallyall 2′-O-methyl adenosine, 2′-O-methyl uridine, 2′-O-methyl cytidine,and 2′-F guanosine. In a preferred embodiment, the resulting modifiedoligonucleotides comprise a sequence where at least 80% of all adenosinenucleotides are 2′-O-methyl adenosine, at least 80% of all uridinenucleotides are 2′-O-methyl uridine, at least 80% of all cytidinenucleotides are 2′-O-methyl cytidine, and at least 80% of all guanosinenucleotides are 2′-F guanosine. In a more preferred embodiment, theresulting modified oligonucleotides comprise a sequence where at least90% of all adenosine nucleotides are 2′-O-methyl adenosine, at least 90%of all uridine nucleotides are 2′-O-methyl uridine, at least 90% of allcytidine nucleotides are 2′-O-methyl cytidine, and at least 90% of allguanosine nucleotides are 2′-F guanosine. The resulting modifiedoligonucleotides comprise a sequence where 100% of all adenosinenucleotides are 2′-O-methyl adenosine, 100% of all uridine nucleotidesare 2′-O-methyl uridine, 100% of all cytidine nucleotides are2′-O-methyl cytidine, and 100% of all guanosine nucleotides are 2′-Fguanosine.

[0134] Under dAmB transcription conditions of the present invention, thetranscription reaction mixture comprises 2′-deoxy adenosinetriphosphates (dATP), 2′-O-methyl cytidine triphosphates (CTP),2′-O-methyl guanosine triphosphates (GTP), and 2′-O-methyl uridinetriphosphates (UTP). The modified oligonucleotides produced using thedAmB transcription mixtures of the present invention comprisesubstantially all 2′-deoxy adenosine, 2′-O-methyl cytidine, 2′-O-methylguanosine, and 2′-O-methyl uridine. In a preferred embodiment, theresulting modified oligonucleotides comprise a sequence where at least80% of all adenosine nucleotides are 2′-deoxy adenosine, at least 80% ofall cytidine nucleotides are 2′-O-methyl cytidine, at least 80% of allguanosine nucleotides are 2′-O-methyl guanosine, and at least 80% of alluridine nucleotides are 2′-O-methyl uridine. In a more preferredembodiment, the resulting modified oligonucleotides comprise a sequencewhere at least 90% of all adenosine nucleotides are 2′-deoxy adenosine,at least 90% of all cytidine nucleotides are 2′-O-methyl cytidine, atleast 90% of all guanosine nucleotides are 2′-O-methyl guanosine, and atleast 90% of all uridine nucleotides are 2′-O-methyl uridine. In a mostpreferred embodiment, the resulting modified oligonucleotides of thepresent invention comprise a sequence where 100% of all adenosinenucleotides are 2′-deoxy adenosine, 100% of all cytidine nucleotides are2′-O-methyl cytidine, 100% of all guanosine nucleotides are 2′-O-methylguanosine, and 100% of all uridine nucleotides are 2′-O-methyl uridine.

[0135] In each case, the transcription products can then be used as thelibrary in the SELEX™ process to identify aptamers and/or to determine aconserved motif of sequences that have binding specificity to a giventarget. The resulting sequences are already stabilized, eliminating thisstep from the process to arrive at a stabilized aptamer sequence andgiving a more highly stabilized aptamer as a result. Another advantageof the 2′-OMe SELEX™ process is that the resulting sequences are likelyto have fewer 2′-OH nucleotides required in the sequence, possibly none.

[0136] As described below, lower but still useful yields of transcriptsfully incorporating 2′-OMe substituted nucleotides can be obtained underconditions other than the optimized conditions described above. Forexample, variations to the above transcription conditions include:

[0137] The HEPES buffer concentration can range from 0 to 1 M. Thepresent invention also contemplates the use of other buffering agentshaving a pKa between 5 and 10, for example without limitation,Tris(hydroxymethyl)aminomethane.

[0138] The DTT concentration can range from 0 to 400 mM. The methods ofthe present invention also provide for the use of other reducing agents,for example without limitation, mercaptoethanol.

[0139] The spermidine and/or spermine concentration can range from 0 to20 mM.

[0140] The PEG-8000 concentration can range from 0 to 50% (w/v). Themethods of the present invention also provide for the use of otherhydrophilic polymer, for example without limitation, other molecularweight PEG or other polyalkylene glycols.

[0141] The Triton X-100 concentration can range from 0 to 0.1% (w/v).The methods of the present invention also provide for the use of othernon-ionic detergents, for example without limitation, other detergents,including other Triton-X detergents.

[0142] The MgCl₂ concentration can range from 0.5 mM to 50 mM. The MnCl₂concentration can range from 0.15 mM to 15 mM. Both MgCl₂ and MnCl₂ mustbe present within the ranges described and in a preferred embodiment arepresent in about a 10 to about 3 ratio of MgCl₂:MnCl₂, preferably, theratio is about 3-5, more preferably, the ratio is about 3 to about 4.

[0143] The 2′-OMe NTP concentration (each NTP) can range from 5 μM to 5mM.

[0144] The 2′-OH GTP concentration can range from 0 μM to 300 μM.

[0145] The 2′-OH GMP concentration can range from 0 to 5 mM.

[0146] The pH can range from pH 6 to pH 9. The methods of the presentinvention can be practiced within the pH range of activity of mostpolymerases that incorporate modified nucleotides. In addition, themethods of the present invention provide for the optional use ofchelating agents in the transcription reaction condition, for examplewithout limitation, EDTA, EGTA, and DTT.

[0147] The selected aptamers having the highest affinity and specificbinding as demonstrated by biological assays as described in theexamples below are suitable therapeutics for treating conditions inwhich the target is involved in pathogenesis.

[0148] Aptamer Therapeutics

[0149] Aptamers represent a promising class of therapeutic agentscurrently in pre-clinical and clinical development. Like biologics,e.g., peptides or monoclonal antibodies, aptamers are capable of bindingspecifically to molecular targets and, through binding, inhibitingtarget function. A typical aptamer is 10-15 kDa in size (i.e., 30-45nucleotides), binds its target with sub-nanomolar affinity, anddiscriminates among closely related targets (e.g., will typically notbind other proteins from the same gene family) (Griffin, et al. (1993),Gene 137(1): 25-31; Jenison, et al. (1998), Antisense Nucleic Acid DrugDev. 8(4): 265-79; Bell, et al. (1999), In Vitro Cell. Dev. Biol. Anim.35(9): 533-42; Watson, et al. (2000), Antisense Nucleic Acid Drug Dev.10(2): 63-75; Daniels, et al. (2002), Anal. Biochem. 305(2): 214-26;Chen, et al. (2003), Proc. Natl. Acad. Sci. U.S.A. 100(16): 9226-31;Khati, et al. (2003), J. Virol. 77(23): 12692-8; Vaish, et al. (2003),Biochemistry 42(29): 8842-51). Created by an entirely in vitro selectionprocess (SELEX™) from libraries of random sequence oligonucleotides,aptamers have been generated against numerous proteins of therapeuticinterest, including growth factors, enzymes, immunoglobulins, andreceptors (Ellington and Szostak (1990), Nature 346(6287): 818-22; Tuerkand Gold (1990), Science 249(4968): 505-510).

[0150] Aptamers have a number of attractive characteristics for use astherapeutics. In addition to high target affinity and specificity,aptamers have shown little or no toxicity or immunogenicity in standardassays (Wlotzka, et al. (2002), Proc. Natl. Acad. Sci. U.S.A. 99(13):8898-902). Several therapeutic aptamers have been optimized and advancedthrough varying stages of pre-clinical development, includingpharmacokinetic analysis, characterization of biological efficacy incellular and animal disease models, and preliminary safety pharmacologyassessment (Reyderman and Stavchansky (1998), Pharmaceutical Research15(6): 904-10; Tucker et al., (1999), J. Chromatography B. 732: 203-212;Watson, et al. (2000), Antisense Nucleic Acid Drug Dev. 10(2): 63-75).

[0151] It is important that the pharmacokinetic properties for alloligonucleotide-based therapeutics, including aptamers, be tailored tomatch the desired pharmaceutical application. While aptamers directedagainst extracellular targets do not suffer from difficulties associatedwith intracellular delivery (as is the case with antisense andRNAi-based therapeutics), such aptamers must be able to be distributedto target organs and tissues, and remain in the body (unmodified) for aperiod of time consistent with the desired dosing regimen. Early work onnucleic acid-based therapeutics has shown that, while unmodifiedoligonucleotides are degraded rapidly by nuclease digestion, protectivemodifications at the 2′-position of the sugar, and use of invertedterminal cap structures, e.g., [3′-3′dT], dramatically improve nucleicacid stability in vitro and in vivo (Green, et al. (1995), Chem. Biol.2(10): 683-95; Jellinek, et al. (1995), Biochemistry 34(36): 11363-72;Ruckman, et al. (1998), J. Biol. Chem. 273(32): 20556-67; Uhlmann, etal. (2000), Methods Enzymol. 313: 268-84). In some SELEX selections(i.e., SELEX experiments or SELEXions), starting pools of nucleic acidsfrom which aptamers are selected are typically pre-stabilized bychemical modification, for example by incorporation of2′-fluoropyrimidine (2′-F) substituted nucleotides, to enhanceresistance of aptamers against nuclease attack. Aptamers incorporating2′-O-methylpurine (2′-OMe purine) substituted nucleotides have also beendeveloped through post-SELEX modification steps or, more recently, byenabling synthesis of 2′-OMe-containing random sequence libraries as anintegral component of the SELEX process itself, as described above.

[0152] In addition to clearance by nucleases, oligonucleotidetherapeutics are subject to elimination via renal filtration. As such, anuclease-resistant oligonucleotide administered intravenously exhibitsan in vivo half-life of <10 min, unless filtration can be blocked. Thiscan be accomplished by either facilitating rapid distribution out of theblood stream into tissues or by increasing the apparent molecular weightof the oligonucleotide above the effective size cut-off for theglomerulus. Conjugation of small therapeutics to a PEG polymer(PEGylation), described below, can dramatically lengthen residence timesof aptamers in circulation, thereby decreasing dosing frequency andenhancing effectiveness against vascular targets. Previous work inanimals has examined the plasma pharmacokinetic properties ofPEG-conjugated aptamers (Reyderman and Stavchansky (1998),Pharmaceutical Research 15(6): 904-10; Watson, et al. (2000), AntisenseNucleic Acid Drug Dev. 10(2): 63-75). Determining the extravasation ofan aptamer therapeutic, including aptamer therapeutics conjugated to amodifying moiety or containing modified nucleotides, and in particular,determining the potential of aptamers or their modified forms to accessdiseased tissues (for example, sites of inflammation, or the interior oftumors) will better define the spectrum of therapeutic opportunities foraptamer intervention.

[0153] The pharmacokinetic profiles of aptamer compositions of theinvention have “tunability” (i.e., the ability to modulate aptamerpharmacokinetics). The tunability of aptamer pharmacokinetics isachieved, for example through conjugation of modifying moieties (e.g.,PEG polymers) to the aptamer and/or the incorporation of modifiednucleotides (e.g., 2′-fluoro and/or 2′-OMe substitutions) to alter thechemical composition of the nucleic acid.

[0154] In addition, the tunability of aptamer pharmacokinetics is usedto modify the biodistribution of an aptamer therapeutic in a subject.For example, in some therapeutic applications, it may be desirable toalter the biodistribution of an aptamer therapeutic in an effort totarget a particular type of tissue or a specific organ (or set oforgans). In these applications, the aptamer therapeutic preferentiallyaccumulates in a specific tissue or organ(s). In other therapeuticapplications, it may be desirable to target tissues displaying acellular marker or a symptom associated with a given disease, cellularinjury or other abnormal pathology, such that the aptamer therapeuticpreferentially accumulates in the affected tissue. For example, asdescribed in copending provisional application U.S. Ser. No. 60/550790,filed on Mar. 5, 2004 and entitled “Controlled Modulation of thePharmacokinetics and Biodistribution of Aptamer Therapeutics”,PEGylation of an aptamer therapeutic (e.g. PEGylation with a 20 kDa PEGpolymer) is used to target inflamed tissues, such that the PEGylatedaptamer therapeutic preferentially accumulates in inflamed tissue.

[0155] The pharmacokinetic and biodistribution profiles of aptamertherapeutics are determined by monitoring a variety of parameters. Suchparameters include, for example, the half-life (t_(1/2)), the plasmaclearance (C1), the volume of distribution (Vss), the area under theconcentration-time curve (AUC), maximum observed serum or plasmaconcentration (C_(max)), and the mean residence time (MRT) of an aptamercomposition. As used herein, the term “AUC” refers to the area under theplot of the plasma concentration of an aptamer therapeutic versus thetime after aptamer administration. The AUC value is used to estimate thebioavailability (i.e., the percentage of administered aptamertherapeutic in the circulation after aptamer administration) and/ortotal clearance (C1) (i.e., the rate at which the aptamer therapeutic isremoved from circulation) of a given aptamer therapeutic. The volume ofdistribution relates the plasma concentration of an aptamer therapeuticto the amount of aptamer present in the body. The larger the Vss, themore an aptamer is found outside of the plasma (i.e., the moreextravasation).

[0156] PDGF and PDGF-VEGF Specific Binding Aptamers as OncologyTherapeutics

[0157] Aptamers specifically capable of binding and inhibiting differentPDGF isoforms are set forth herein. These aptamers, which includeaptamers that bind only to PDGF, aptamers that bind to both PDGF andVEGF, and either of the above aptamers having a CpG motif incorporatedtherein, provide a low-toxicity, safe, and effective modality ofinhibiting most PDGF-mediated tumor progression, including withoutlimitation, glioblastomas, chronic myelomonocytic leukemia (CMML),dermatofibrosarcoma protuberans (DFSP), gastrointestinal stromal tumors,(GIST), and other soft tissue sarcomas.

[0158] Examples of PDGF and PDGF-VEGF specific binding aptamers for useas oncology therapeutics include the following sequences:

[0159] PDGF-Binding Aptamers: ARC126:5′-(5′-NH2-dC-dA-dG-dG-dC-fU-dA-fC-mG- 3′, SEQ ID No. 1)-HEG-(5′-dC-dG-T-dA-mG-dA-mG-dC-dA-fU-fC-mA-3′, SEQ ID No. 2)-HEG-(5′-T-dG-dA-T-fC-fC-fU-mG-3′dT-3′, SEQ ID No. 3)- 3′

[0160] wherein HEG=hexaethylene glycol amidite. ARC127: 5′-[40KPEG]-(5′-NH2-dC-dA-dG-dG-dC-fU-dA-fC-mG- 3′, SEQ ID No. 1)-HEG-(5′-dC-dG-T-dA-mG-dA-mG-dC-dA-fU-fC-mA-3′, SEQ ID No. 2)-HEG-(5′-T-dG-dA-T-fC-fC-fU-mG-3′dT-3′, SEQ ID No. 3)- 3′

[0161] wherein HEG=hexaethylene glycol amidite. ARC240: 5′-[20KPEG]-(5′-NH2-dC-dA-dG-dG-dC-fU-dA-fC-mG- 3′, SEQ ID No. 1)-HEG-(5′-dC-dG-T-dA-mG-dA-mG-dC-dA-fU-fC-mA-3′, SEQ ID No. 2)-HEG-(5′-T-dG-dA-T-fC-fC-fU-mG-3′dT-3′, SEQ ID No. 3)- 3′

[0162] wherein HEG=hexaethylene glycol amidite. ARC308: 5′-[30KPEG]-(5′-NH2-dC-dA-dG-dG-dC-fU-dA-fC-mG- 3′, SEQ ID No. 1)-HEG-(5′-dC-dG-T-dA-mG-dA-mG-dC-dA-fU-fC-mA-3′, SEQ ID No. 2)-HEG-(5′-T-dG-dA-T-fC-fC-fU-mG-3′dT-3′, SEQ ID No. 3)-3′

[0163] wherein HEG=hexaethylene glycol amidite. deoxyARC126:5′-dCdAdGdGdCdTdAdCdGdCdGdTdAdGdAdGdCdAdTdCdAdTdGdAdTdCdCdTdG-[3T]-3′(SEQ ID NO: 8)

[0164] wherein “d” indicates unmodified deoxynucleotides and“[3T]” is asdefined above. ARC124: 5′ CACAGGCTACGGCACGTAGAGCATCACCATGATCCTGTG3′InvdT(SEQ ID NO.: 11)

[0165] Scrambled Control Aptamer: ARC128: (Scrambled ARC126):5′-(5′-NH2-dC-dA-dG-fC-mG-fU-dA-fC-mG-3′, SEQ ID No. 4)-HEG-(5′-dC-dG-T-dA-dC-dC-mG-dA-T-fU-fC-mA-3′, SEQ ID No. 5)-HEG-(5′-T-dG-dA-dA-dG-fC-fU-mG-3′dT-3′, SEQ ID No. 6)- 3′

[0166] wherein HEG=hexaethylene glycol amidite.

[0167] VEGF-Binding Aptamer: ARC245:5′-mAmUmGmCmAmGmUmUmUmGmAmGmAmAmGmUmCmGmCmGmCmAmU- [3T]-3′, (SEQ ID NO:7)

[0168] wherein “m” indicates 2′-OMe nucleotides and “[3T]”refers to aninverted thymidine nucleotide that is attached to the 3′end of theoligonucleotide at the 3′ position on the ribose sugar, thus theoligonucleotide has two 5′ends and is thus resistant to nucleases actingon the 3′ hydroxyl end.

[0169] PDGF/VEGF Binding Multivalent Aptamers: TK.131.012.A: (SEQ ID NO:9) 5′dCdAdGdGdCdTdAdCdGmAmUmGmCmAmGmUmUmUmGmAmGmAmAmGmUmCmGmCmGmCmAmUdCdGdTdAdGdAdGdCdAdTdCdAdGdAdAdAdTdGdAdTdCdCdTdG[3T]- 3′,

[0170] wherein “m” indicates 2′-OMe nucleotides, “d” and “[3T]”are asdefined above. TK.131.012.B: (SEQ ID NO: 10)5′dCdAdGdGdCdTdAdCdGmUmGmCmAmGmUmUmUmGmAmGmAmAmGmUmCmGmCmGmCmAdCdGdTdAdGdAdGdCdAdTdCdAdGdAdAdAdTdGdAdTdCdCdTdG-[3T]

[0171] wherein “m” and “[3T]” are as defined above.

[0172] Other aptamers that bind PDGF and/or VEGF are described below,e.g., in Table 2 and in Example 12.

[0173] It has been demonstrated that inhibition of PDGF signaling withsmall molecule receptor antagonists decreases interstitial fluidpressure and increases the uptake of chemotherapeutics into solidtumors. Pietras et al. validated the hypothesis that PDGF-B is involvedin IFP and that blocking PDGF-B function could lead to increased uptakeof chemotherapeutics into tumors. (Pietras et al., (2003), Cancer Cellvol. 3 p.439-443). Using the KAT-4 thyroid carcinoma model, which hasknown PDGF paracrine signaling properties, Pietras et al. demonstratedthat KAT-4 tumors expressed PDGF β-receptors in the stroma and thatPDGF-B bound KAT-4 cells in vitro. Next, Pietras et al. used thetyrosine kinase inhibiting drug STI571 (GLEEVEC™) to block PDGF-Bsignaling in KAT-4 tumors and showed that this treatment significantlydecreased tumor IFP in vivo leading to increased uptake of taxol.However, since STI571 targets both PDGF α and β receptors, as well asKit, Abl, and Arg tyrosine kinases, it was impossible to know if theeffect of STI571 was due to PDGF-B blockage alone. This ambiguity wassolved by using a highly specific aptamer to block PDGF-B in similarexperiments. The aptamer has an affinity of 100 pM for PDGF-B and noappreciable affinity for the PDGF-A sequence. As with STI571, treatmentof KAT-4 xenograft mice with PEG-conjugated PDGF-B aptamer lowered IFPand dramatically increased tumor uptake of taxol. Most importantly,aptamer treatment strongly enhanced taxol's ability to inhibit tumorgrowth. In addition, a currently marketed cancer therapeutic, thePDGF-receptor antagonist GLEEVEC™ has been shown to be effective atreducing tumor IFP and increasing the tumor uptake of cytotoxins whenused in combination with a cytotoxin such as taxol. Thus, the methodsand materials of the present are used to inhibit the biological activityof PDGF-B and its etiology in tumor development and growth by enhancingthe uptake and efficacy of chemotherapeutics.

[0174] In other words, combination therapy methods of the presentinvention combining PDGF-specific aptamers of the present invention withcytotoxic agents, i.e. combining PDGF-specific aptamers with other knowncytotoxins, provides an effective method of delivering drug specificallyat the site of tumors, by the selective lowering of IFP in tumorvessels, which in turn allows the increased uptake of cytotoxins intumors through the tumor vasculature. FIG. 4 shows a schematic of thetransport to cytotoxins across tumor vasculature with and without PDGFantagonists by the methods of the present invention (Pietras et al.,(2003), Cancer Cell vol. 3 p.439-443).

[0175] The PDGF aptamers of the present invention can be used incombination with a variety of known cytotoxic or cytostatic(collectively, “cytotoxic”) agents to lower tumor IFP, and therebyincrease delivery and tumor uptake of cytotoxic agents to all solidtumors. Suitable cytotoxic or cytostatic agents include tubulinstabilizers/destabilizers, anti-metabolites, purine synthesisinhibitors, nucleoside analogs, and DNA alkylating or other DNAmodifying agents, including but not limited to paclitaxel (TAXOL™),docetaxel, irinotecan, topotecan, gemcitabine, epothilone, cisplatinum,carboplatin, 5-fluoro-U, calicheamycin, doxorubicin, methotrexate, AraC(cytarabine), vinblastin, daunorubicin, oxaliplatinum, cyclophosphamide,iflosamide, pharmarubicin, epirubicin, vinflunine, oblimersen sodium,permetrexed, kinase inhibitors, including but not limited to EGFreceptor kinase, VEGF receptor kinase, aurora kinase, either alone or inany combination thereof. The PDGF aptamers of the present invention canbe used in combination with a variety of known vascular targeting agentswherein “vascular targeting agent” means a small molecule therapeutic(e.g., irenotecan), a protein therapeutic (e.g., bevicizumab), and/or anoligonucleotide therapeutic (e.g., antisense molecules, siRNA molecules)which modifies the existing vasculature and neovasculature network thatsupports blood and lymphatic flow to the tumor. The PDGF aptamers of thepresent invention can be used in combination with a conjugatetherapeutic comprising a binding or targeting moiety, and a cytotoxicmoiety, wherein the binding or targeting moiety is, but is not limitedto, an aptamer, antibody, including, but not limited to trastuzumab,rituximab, cetuximab, panitumumab, gemtuzumab, bevicizumab, andtositumomab, peptide, a vascular targeting agent or folate compound, andwherein the cytotoxic moiety belongs to a class of compounds includingbut not limited to, tubulin stabilizers/destabilizers, anti-metabolites,purine synthesis inhibitors, nucleoside analogs, and DNA alkylatingagents, other DNA modifying agents, or vascular disruptive agents (e.g.,flavonoids) alone or in any combination thereof. The materials andmethods of the present invention provide a more effective way ofadministering known chemotherapeutic agents to inhibit solid tumorformation which results in a variety of cancers including but notlimited to colorectal, pancreatic, breast, lung, prostate, and ovariancancer.

[0176] In addition, PDGF aptamer compositions of the present inventioncan be used as anti-angiogenic agents to inhibit the formation of tumorvasculature by targeting pericytes. Alternatively, PDGF aptamercompositions of the present invention can be used in combination with aVEGF/VEGFR antagonist, such as an aptamer specific for VEGF, to providea more effective way of inhibiting tumor angiogenesis than either a PDGFaptamer therapeutic or VEGF/VEGFR antagonists therapeutic alone. Anadvantage of the PDGF-B- and VEGF-targeting agents of the presentinvention is that the therapeutic compositions of the present inventiondo not appear to exhibit any off-target tyrosine kinase activity, sincethey are exquisitely specific to their target ligand, and are notdesigned to enter cells to elicit their biological function. Incontrast, currently marketed small molecule kinase inhibitortherapeutics, for example GLEEVEC™, exhibit high off-target activity.GLEEVEC™ targets both the α and β tyrosine kinase receptors in additionto BCR-Abl, C-kit and Arg tyrosine kinases. Small molecule kinaseinhibitors, such as GLEEVEC™, are administered at or near their maximumtolerated dose when used in the treatment of solid tumors. The toxicityof GLEEVEC™ and other tyrosine kinase inhibitors (TKIs) is likelylimited by both mechanism-related side-effects and non-mechanism(off-target) related side-effects. Hence, off-target activity is a majorimpediment to the development of small molecule kinase inhibitors ingeneral. Based on in vivo experiments with ARC127 (5′-[40K PEG]-(SEQ IDNo.1)-HEG-(SEQ ID NO:2)-HEG-(SEQ ID NO:3)-3′-dT-3′) and ARC308 (5′-[30KPEG]-(SEQ ID No.1)-HEG-(SEQ ID NO:2)-HEG-(SEQ ID NO:3)-3′-dT-3′)dose-limiting side effects are not evident. Additional advantages arethat the aptamer therapeutic compositions of the present invention canbe administered via intravenous, subcutaneous, or intra-peritonealroutes, which means ease of administration. When compared to monoclonalantibody cancer therapeutics, aptamers are non-immunogenic, thusreaction to the drug or resistance to the drug are not an issue.

[0177] Aptamers to PDGF and PDGF Isoforms

[0178] The materials of the present invention comprise a series ofnucleic acid aptamers of 31-35 nucleotides in length (SEQ ID NO:1 to SEQID NO:3, SEQ ID NO:9 to SEQ ID NO:38, SEQ ID NO:50, SEQ ID NO:54 to SEQID NO:90, and SEQ ID NO:94 to SEQ ID NO:99) which bind specifically toPDGF-B protein in vitro and which functionally block the activity ofPDGF-BB in in vivo and cell-based assays. The anti-PDGF-B aptamerssequences of the present invention are derived from a parent moleculeARC126 (5′-(SEQ ID No.1)-HEG-(SEQ ID NO:2)-HEG-(SEQ ID NO:3)-3′-dT-3′)which contains seven individual 2′F containing residues. 2′F containingresidues were incorporated into ARC126 to increase the in vitro serumand in vivo stability of the therapeutic aptamer by blocking itsdegradation by serum endonucleases and/or exonucleases. In an effort toreplace potentially toxic 2′F containing nucleotide residues in theARC126 anti-PDGF-B aptamer, a new series of fully 2′F-free aptamers havebeen identified. The new aptamers of the present invention retain potentin vitro binding and anti-proliferative activity, and contain naturallyoccurring 2′deoxy or 2′OMe substituted nucleotides. These new aptamersof the present invention also retain substantial serum stability asdetermined through resistance to nuclease degradation in an in vitrostability assay, with no degradation detected for up to 48 hours.

[0179] The aptamer therapeutics of the present invention have greataffinity and specificity to PDGF, PDGF isoforms, and PDGF receptor whilereducing the deleterious side effects from non-naturally occurringnucleotide substitutions when the aptamer therapeutics break down in thebody of patients or subjects. The therapeutic compositions containingthe aptamer therapeutics of the present invention are free of or have areduced amount of fluorinated nucleotides.

[0180] Materials and Methods to Increase the Efficacy of Anti-TumorAgents

[0181] The materials and methods of the present invention furthercomprise methods to increase the efficacy of antitumor agents by dualtherapy with the aptamers of the present invention, namely ARC127 andARC308. In addition, the methods of the present invention havedemonstrated that PDGF-B specific aptamers, ARC127 (i.e., ARC126+40KPEG) and ARC308 (i.e., ARC 126+30K PEG) are active anti-tumor agentswhen co-administered with irinotecan to nude mice bearing the colorectalLS174t tumor xenograft. The use of the cancer therapeutic methods of thepresent invention have shown that both ARC127 and ARC308 are safenon-cytotoxic agents when administered alone, but in combination withother cytotoxic agents, ARC127 and ARC308 potentiate their anti-tumoreffects through a novel mechanism of action. The methods of the presentinvention further demonstrate that the serum-derived ARC127 aptamer whenadministered to mice parenterally, i.e. intravenously, subcutaneously,or by intraperitoneal injection, retains full biological activity.

[0182] Aptamer-Chimera Specific for PDGF-B and VEGF

[0183] The materials and methods of the present invention furtherprovide bi-functional aptamer-chimera that target both PDGF-B and VEGF.The PDGF-B-VEGF aptamer chimera TK.131.12.A (SEQ ID No.9) andTK.131.12.B (SEQ ID No.10) of the present invention allow for thesimultaneous targeting of PDGF-B and VEGF for the treatment of cancer.The PDGF-B aptamer used in the chimeric molecule is derived from theARC127 aptamer sequence. The VEGF aptamer that was used in the chimericmolecule is derived from the ARC245 (SEQ ID No.7) aptamer sequence. Theaptamer-chimera of the present invention can be constructed from any setof PDGF-B and VEGF binding aptamers. The PDGF-B-VEGF chimera of thepresent invention is useful in the treatment of VEGF-dependent solidtumors that also show a high degree of neovascularization as well aspericyte recruitment to support the nascent vasculature.

[0184] Recent anti-tumor data from the RipTag pancreatic mouse tumormodel suggests that there is a greater block in tumor growth conferredwhen anti-VEGF and anti-PDGFR therapy are undertaken simultaneously,than when either the anti-VEGF agent or the anti-PDGFR agent is addedalone (Bergers et al., (2003), J. Clin. Invest., 111:9, p. 1287-1295).Since, anti-PDGFR therapy blocks all receptor-mediated signaling events,its effects can be expected to be non-specific. The PDGF-B-VEGF chimerain this invention provide for precise PDGF-B and VEGF targeting intumors.

[0185] The aptamer therapeutics of the present invention have greataffinity and specificity to VEGF, and VEGF receptor while reducing thedeleterious side effects from non-naturally occurring nucleotidesubstitutions when the aptamer therapeutics break down in the body ofpatients or subjects. The therapeutic compositions of the aptamertherapeutics of the present invention are free of or have a reducedamount of fluorinated nucleotides.

[0186] Aptamers Having Immunostimulatory Motifs

[0187] Recognition of bacterial DNA by the vertebrate immune system isbased on the recognition of unmethylated CG dinucleotides in particularsequence contexts (“CpG motifs”). One receptor that recognizes such amotif is Toll-like receptor 9 (“TLR 9”), a member of a family ofToll-like receptors (˜10 members) that participate in the innate immuneresponse by recognizing distinct microbial components. TLR 9 bindsunmethylated oligodeoxynucleotide (ODN) CpG sequences in asequence-specific manner. The recognition of CpG motifs triggers defensemechanisms leading to innate and ultimately acquired immune responses.For example, activation of TLR 9 in mice induces activation of antigenpresenting cells, up regulation of MHC class I and II molecules andexpression of important costimulatory molecules and cytokines includingIL-12 and IL-23. This activation both directly and indirectly enhances Band T cell responses, including robust up regulation of the TH1 cytokineIFN-gamma. Collectively, the response to CpG sequences leads to:protection against infectious diseases, improved immune response tovaccines, an effective response against asthma, and improvedantibody-dependent cell-mediated cytotoxicity. Thus, CpG ODN's canprovide protection against infectious diseases, function asimmuno-adjuvants or cancer therapeutics (monotherapy or in combinationwith mAb or other therapies), and can decrease asthma and allergicresponse.

[0188] Aptamers comprising one or more CpG motifs can be identified orgenerated by a variety of strategies using, e.g., the SELEX™ processdescribed herein. In general the strategies can be divided into twogroups. In the first group, the strategies are directed to identifyingor generating aptamers comprising both a binding site for targets otherthan those recognizing CpG motifs and a CpG motif These strategies areas follows: (a) performing SELEX™ to obtain an aptamer to a specifictarget (other than a target known to bind to CpG motifs and upon bindingstimulate an immune response), preferably a target where a repressedimmune response is relevant to disease development, using anoligonucleotide pool wherein a CpG motif has been incorporated into eachmember of the pool as, or as part of, a fixed region, e.g., in therandomized region of the pool members; (b) performing SELEX to obtain anaptamer to a specific target (other than a target known to bind to CpGmotifs and upon binding stimulate an immune response), preferably atarget where a repressed immune response is relevant to diseasedevelopment, and then appending a CpG motif to the 5′ and/or 3′ end orengineering a CpG motif into a region, preferably a non-essentialregion, of the aptamer; (c) performing SELEX™ to obtain an aptamer to aspecific target (other than a target known to bind to CpG motifs andupon binding stimulate an immune response), preferably a target where arepressed immune response is relevant to disease development, whereinduring synthesis of the pool the molar ratio of the various nucleotidesis biased in one or more nucleotide addition steps so that therandomized region of each member of the pool is enriched in CpG motifs;and (d) performing SELEX™ to obtain an aptamer to a specific target(other than a target known to bind to CpG motifs and upon bindingstimulate an immune response), preferably a target where a repressedimmune response is relevant to disease development, and identifyingthose aptamers comprising a CpG motif.

[0189] In the second group, the strategies are directed to identifyingor generating aptamers comprising a CpG motif and/or other sequencesthat are bound by the receptors for the CpG motifs (e.g., TLR9 or theother toll-like receptors) and upon binding stimulate an immuneresponse. These strategies are as follows: (i) performing SELEX™ toobtain an aptamer to a target known to bind to CpG motifs and uponbinding stimulate an immune response using an oligonucleotide poolwherein a CpG motif has been incorporated into each member of the poolas, or as part of, a fixed region, e.g., in the randomized region of thepool members; (ii) performing SELEX to obtain an aptamer to a targetknown to bind to CpG motifs and upon binding stimulate an immuneresponse and then appending a CpG motif to the 5′ and/or 3′ end orengineering a CpG motif into a region, preferably a non-essentialregion, of the aptamer; (iii) performing SELEX to obtain an aptamer to atarget known to bind to CpG motifs and upon binding stimulate an immuneresponse wherein during synthesis of the pool, the molar ratio of thevarious nucleotides is biased in one or more nucleotide addition stepsso that the randomized region of each member of the pool is enriched inCpG motifs ; (iv) performing SELEX to obtain an aptamer to a targetknown to bind to CpG motifs and upon binding stimulate an immuneresponse and identifying those aptamers comprising a CpG motif; and (v)performing SELEX to obtain an aptamer to a target known to bind to CpGmotifs and identifying those aptamers which, upon binding, stimulate animmune response not comprising a CpG motif.

[0190] A variety of different classes of CpG motifs have beenidentified, each resulting upon recognition in a different cascade ofevents, release of cytokines and other molecules, and activation ofcertain cell types. See, e.g., CpG Motifs in Bacterial DNA and TheirImmune Effects, Annu. Rev. Immunol. 2002, 20:709-760, incorporatedherein by reference. Additional immunostimulatory motifs are disclosedin the following U.S. Patents, each of which is incorporated herein byreference: U.S. Pat. Nos. 6,207,646; 6,239,116; 6,429,199; 6,214,806;6,653,292; 6,426,434; 6,514,948 and 6,498,148. Any of these CpG or otherimmunostimulatory motifs can be incorporated into an aptamer. The choiceof aptamers is dependent on the disease or disorder to be treated.Preferred immunostimulatory motifs are as follows (shown the 5′ to 3′left to right) wherein “r” designates a purine, “y” designates apyrimidine, and “X” designates any nucleotide: AACGTTCGAG (SEQ IDNO:37); AACGTT; ACGT, rCGy; rrCGyy, XCGX, XXCGXX, and X₁X₂CGY₁Y₂ whereinX₁ is G or A, X₂ is not C, Y₁ is not G and Y₂ is preferably T.

[0191] In those instances where a CpG motif is incorporated into anaptamer that binds to a specific target other than a target known tobind to CpG motifs and upon binding stimulate an immune response (a“non-CpG target”), the CpG is preferably located in a non-essentialregion of the aptamer. Non-essential regions of aptamers can beidentified by site-directed mutagenesis, deletion analyses and/orsubstitution analyses. However, any location that does not significantlyinterfere with the ability of the aptamer to bind to the non-CpG targetmay be used. In addition to being embedded within the aptamer sequence,the CpG motif may be appended to either or both of the 5′ and 3′ ends orotherwise attached to the aptamer. Any location or means of attachmentmay be used so long as the ability of the aptamer to bind to the non-CpGtarget is not significantly interfered with.

[0192] As used herein, “stimulation of an immune response” can meaneither (1) the induction of a specific response (e.g., induction of aTh1 response) or of the production of certain molecules or (2) theinhibition or suppression of a specific response (e.g., inhibition orsuppression of the Th2 response) or of certain molecules.

[0193] CpG motifs can be incorporated or appended to an aptamer againstany target including, but not limited to: PDGF, IgE, IgE Fcε R1, TNFa,PSMA, CTLA4, PD-1, PD-L1, PD-L2, FcRIIB, BTLA, TIM-3, CD11b, CD-11c,BAFF, B7-X, CD19, CD20, CD25 and CD33.

[0194] By incorporating CpG motifs into aptamers specifically targetingsolid tumors these aptamers can be used to activate the immune systemthrough the recruitment of antigen presenting cells that have taken uptumor derived material, enhance their maturation and migration to locallymph nodes and increase priming of tumor specific T-cells. This isespecially relevant where aptamers deliver cytotoxic payload and resultin cell death (such as a PSMA aptamer containing a CpG motif). Such CpGmotif containing aptamers can also induce tumor-specific memory response(prophylactic use). In addition, the IFP lowering and pericyterecruitment blocking effects of a PDGF-B aptamer combined with theincreased immune response observed upon CpG administration represents apotent therapeutic for cancer. Thus, aptamers with incorporated,appended or embedded CpG motifs represent a novel class of anti-cancercompounds such that when administered they can lead to a significantde-bulking of the tumor through two mechanisms: first, throughactivation of tumor specific T-cells within the tumor bed and second,through the intended mechanism-based action of the aptamerpharmacophore.

[0195] Pharmaceutical Compositions

[0196] The invention also includes pharmaceutical compositionscontaining aptamer molecules. In some embodiments, the compositions aresuitable for internal use and include an effective amount of apharmacologically active compound of the invention, alone or incombination, with one or more pharmaceutically acceptable carriers. Thecompounds are especially useful in that they have very low, if anytoxicity.

[0197] Compositions of the invention can be used to treat or prevent apathology, such as a disease or disorder, or alleviate the symptoms ofsuch disease or disorder in a patient. Compositions of the invention areuseful for administration to a subject suffering from, or predisposedto, a disease or disorder which is related to or derived from a targetto which the aptamers specifically bind.

[0198] For example, the target is a protein involved with a pathology,for example, the target protein causes the pathology. For example, thetarget is PDGF involvement in the development and progression of solidtumors.

[0199] Compositions of the invention can be used in a method fortreating a patient or subject having a pathology. The method involvesadministering to the patient or subject a composition comprisingaptamers that bind a target (e.g., a protein) involved with thepathology, so that binding of the composition to the target alters thebiological function of the target, thereby treating the pathology.

[0200] The patient or subject having a pathology, e.g. the patient orsubject treated by the methods of this invention can be a mammal, ormore particularly, a human.

[0201] In practice, the compounds or their pharmaceutically acceptablesalts, are administered in amounts which will be sufficient to exerttheir desired biological activity, e.g., inhibiting the binding of acytokine to its receptor.

[0202] One aspect of the invention comprises an aptamer composition ofthe invention in combination with other treatments for cancer or cancerrelated disorders. The aptamer composition of the invention may contain,for example, more than one aptamer. In some examples, an aptamercomposition of the invention, containing one or more compounds of theinvention, is administered in combination with another usefulcomposition such as an anti-inflammatory agent, an immunosuppressant, anantiviral agent, or the like. Furthermore, the compounds of theinvention may be administered in combination with a cytotoxic,cytostatic, or chemotherapeutic agent such as an alkylating agent,anti-metabolite, mitotic inhibitor or cytotoxic antibiotic, as describedabove. In general, the currently available dosage forms of the knowntherapeutic agents for use in such combinations will be suitable.

[0203] “Combination therapy” (or “co-therapy”) includes theadministration of an aptamer composition of the invention and at least asecond agent as part of a specific treatment regimen intended to providethe beneficial effect from the co-action of these therapeutic agents.The beneficial effect of the combination includes, but is not limitedto, pharmacokinetic or pharmacodynamic co-action resulting from thecombination of therapeutic agents. Administration of these therapeuticagents in combination typically is carried out over a defined timeperiod (usually minutes, hours, days or weeks depending upon thecombination selected).

[0204] “Combination therapy” may, but generally is not, intended toencompass the administration of two or more of these therapeutic agentsas part of separate monotherapy regimens that incidentally andarbitrarily result in the combinations of the present invention.“Combination therapy” is intended to embrace administration of thesetherapeutic agents in a sequential manner, that is, wherein eachtherapeutic agent is administered at a different time, as well asadministration of these therapeutic agents, or at least two of thetherapeutic agents, in a substantially simultaneous manner.Substantially simultaneous administration can be accomplished, forexample, by administering to the subject a single capsule having a fixedratio of each therapeutic agent or in multiple, single capsules for eachof the therapeutic agents.

[0205] Sequential or substantially simultaneous administration of eachtherapeutic agent can be effected by any appropriate route including,but not limited to, topical routes, oral routes, intravenous routes,intramuscular routes, and direct absorption through mucous membranetissues. The therapeutic agents can be administered by the same route orby different routes. For example, a first therapeutic agent of thecombination selected may be administered by injection while the othertherapeutic agents of the combination may be administered topically.

[0206] Alternatively, for example, all therapeutic agents may beadministered topically or all therapeutic agents may be administered byinjection. The sequence in which the therapeutic agents are administeredis not narrowly critical. “Combination therapy” also can embrace theadministration of the therapeutic agents as described above in furthercombination with other biologically active ingredients. Where thecombination therapy further comprises a non-drug treatment, the non-drugtreatment may be conducted at any suitable time so long as a beneficialeffect from the co-action of the combination of the therapeutic agentsand non-drug treatment is achieved. For example, in appropriate cases,the beneficial effect is still achieved when the non-drug treatment istemporally removed from the administration of the therapeutic agents,perhaps by days or even weeks.

[0207] The compounds of the invention and the other pharmacologicallyactive agent may be administered to a patient simultaneously,sequentially or in combination. It will be appreciated that when using acombination of the invention, the compound of the invention and theother pharmacologically active agent may be in the same pharmaceuticallyacceptable carrier and therefore administered simultaneously. They maybe in separate pharmaceutical carriers such as conventional oral dosageforms which are taken simultaneously. The term “combination” furtherrefers to the case where the compounds are provided in separate dosageforms and are administered sequentially.

[0208] Therapeutic or pharmacological compositions of the presentinvention will generally comprise an effective amount of the activecomponent(s) of the therapy, dissolved or dispersed in apharmaceutically acceptable medium. Pharmaceutically acceptable media orcarriers include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like. The use of such media and agents for pharmaceuticalactive substances is well known in the art. Supplementary activeingredients can also be incorporated into the therapeutic compositionsof the present invention.

[0209] The preparation of pharmaceutical or pharmacological compositionswill be known to those of skill in the art in light of the presentdisclosure. Typically, such compositions may be prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid prior to injection; as tablets orother solids for oral administration; as time release capsules; or inany other form currently used, including eye drops, creams, lotions,salves, inhalants and the like. The use of sterile formulations, such assaline-based washes, by surgeons, physicians or health care workers totreat a particular area in the operating field may also be particularlyuseful. Compositions may also be delivered via microdevice,microparticle or sponge.

[0210] Upon formulation, therapeutics will be administered in a mannercompatible with the dosage formulation, and in such amount as ispharmacologically effective. The formulations are easily administered ina variety of dosage forms, such as the type of injectable solutionsdescribed above, but drug release capsules and the like can also beemployed.

[0211] In this context, the quantity of active ingredient and volume ofcomposition to be administered depends on the host animal to be treated.Precise amounts of active compound required for administration depend onthe judgment of the practitioner and are peculiar to each individual.

[0212] A minimal volume of a composition required to disperse the activecompounds is typically utilized. Suitable regimes for administration arealso variable, but would be typified by initially administering thecompound and monitoring the results and then giving further controlleddoses at further intervals.

[0213] For instance, for oral administration in the form of a tablet orcapsule (e.g., a gelatin capsule), the active drug component can becombined with an oral, non-toxic pharmaceutically acceptable inertcarrier such as ethanol, glycerol, water and the like. Moreover, whendesired or necessary, suitable binders, lubricants, disintegratingagents and coloring agents can also be incorporated into the mixture.Suitable binders include starch, magnesium aluminum silicate, starchpaste, gelatin, methylcellulose, sodium carboxymethylcellulose and/orpolyvinylpyrrolidone, natural sugars such as glucose or beta-lactose,corn sweeteners, natural and synthetic gums such as acacia, tragacanthor sodium alginate, polyethylene glycol, waxes and the like. Lubricantsused in these dosage forms include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride,silica, talcum, stearic acid, its magnesium or calcium salt and/orpolyethyleneglycol and the like. Disintegrators include, withoutlimitation, starch, methyl cellulose, agar, bentonite, xanthan gumstarches, agar, alginic acid or its sodium salt, or effervescentmixtures, and the like. Diluents, include, e.g., lactose, dextrose,sucrose, mannitol, sorbitol, cellulose and/or glycine.

[0214] Injectable compositions are preferably aqueous isotonic solutionsor suspensions, and suppositories are advantageously prepared from fattyemulsions or suspensions. The compositions may be sterilized and/orcontain adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure and/or buffers. In addition, they may also contain othertherapeutically valuable substances. The compositions are preparedaccording to conventional mixing, granulating or coating methods,respectively, and contain about 0.1 to 75%, preferably about 1 to 50%,of the active ingredient.

[0215] The compounds of the invention can also be administered in suchoral dosage forms as timed release and sustained release tablets orcapsules, pills, powders, granules, elixirs, tinctures, suspensions,syrups and emulsions.

[0216] Liquid, particularly injectable compositions can, for example, beprepared by dissolving, dispersing, etc. The active compound isdissolved in or mixed with a pharmaceutically pure solvent such as, forexample, water, saline, aqueous dextrose, glycerol, ethanol, and thelike, to thereby form the injectable solution or suspension.Additionally, solid forms suitable for dissolving in liquid prior toinjection can be formulated. Injectable compositions are preferablyaqueous isotonic solutions or suspensions. The compositions may besterilized and/or contain adjuvants, such as preserving, stabilizing,wetting or emulsifying agents, solution promoters, salts for regulatingthe osmotic pressure and/or buffers. In addition, they may also containother therapeutically valuable substances.

[0217] The compounds of the present invention can be administered inintravenous (both bolus and infusion), intraperitoneal, subcutaneous orintramuscular form, all using forms well known to those of ordinaryskill in the pharmaceutical arts. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions.

[0218] Parenteral injectable administration is generally used forsubcutaneous, intramuscular or intravenous injections and infusions.Additionally, one approach for parenteral administration employs theimplantation of a slow-release or sustained-released systems, whichassures that a constant level of dosage is maintained, according to U.S.Pat. No. 3,710,795, incorporated herein by reference.

[0219] Furthermore, preferred compounds for the present invention can beadministered in intranasal form via topical use of suitable intranasalvehicles, or via transdermal routes, using those forms of transdermalskin patches well known to those of ordinary skill in that art. To beadministered in the form of a transdermal delivery system, the dosageadministration will, of course, be continuous rather than intermittentthroughout the dosage regimen. Other preferred topical preparationsinclude creams, ointments, lotions, aerosol sprays and gels, wherein theconcentration of active ingredient would range from 0.01% to 15%, w/w orw/v.

[0220] For solid compositions, excipients include pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharin,talcum, cellulose, glucose, sucrose, magnesium carbonate, and the likemay be used. The active compound defined above, may be also formulatedas suppositories using for example, polyalkylene glycols, for example,propylene glycol, as the carrier. In some embodiments, suppositories areadvantageously prepared from fatty emulsions or suspensions.

[0221] The compounds of the present invention can also be administeredin the form of liposome delivery systems, such as small unilamellarvesicles, large unilamellar vesicles and multilamellar vesicles.Liposomes can be formed from a variety of phospholipids, containingcholesterol, stearylamine or phosphatidylcholines. In some embodiments,a film of lipid components is hydrated with an aqueous solution of drugto a form lipid layer encapsulating the drug, as described in U.S. Pat.No. 5,262,564. For example, the aptamer molecules described herein canbe provided as a complex with a lipophilic compound or non-immunogenic,high molecular weight compound constructed using methods known in theart. An example of nucleic-acid associated complexes is provided in U.S.Pat. No. 6,011,020.

[0222] The compounds of the present invention may also be coupled withsoluble polymers as targetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxypropyl-methacrylamide-phenol,polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds of thepresent invention may be coupled to a class of biodegradable polymersuseful in achieving controlled release of a drug, for example,polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates andcross-linked or amphipathic block copolymers of hydrogels.

[0223] If desired, the pharmaceutical composition to be administered mayalso contain minor amounts of non-toxic auxiliary substances such aswetting or emulsifying agents, pH buffering agents, and other substancessuch as for example, sodium acetate, and triethanolamine oleate.

[0224] The dosage regimen utilizing the compounds is selected inaccordance with a variety of factors including type, species, age,weight, sex and medical condition of the patient; the severity of thecondition to be treated; the route of administration; the renal andhepatic function of the patient; and the particular compound or saltthereof employed. An ordinarily skilled physician or veterinarian canreadily determine and prescribe the effective amount of the drugrequired to prevent, counter or arrest the progress of the condition.

[0225] Oral dosages of the present invention, when used for theindicated effects, will range between about 0.05 to 5000 mg/day orally.The compositions are preferably provided in the form of scored tabletscontaining 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100.0, 250.0,500.0 and 1000.0 mg of active ingredient. Effective plasma levels of thecompounds of the present invention range from 0.002 mg to 50 mg per kgof body weight per day.

[0226] Compounds of the present invention may be administered in asingle daily dose, or the total daily dosage may be administered individed doses of two, three or four times daily.

[0227] Modulation of Pharmacokinetics and Biodistribution of AptamerTherapeutics

[0228] The present invention provides materials and methods to affectthe pharmacokinetics of aptamer compositions, and, in particular, theability to tune (i.e., the “tunability”) aptamer pharmacokinetics. Thetunability of aptamer pharmacokinetics is achieved through conjugationof modifying moieties to the aptamer and/or the incorporation ofmodified nucleotides to alter the chemical composition of the nucleicacid. The ability to tune aptamer pharmacokinetics is used in theimprovement of existing therapeutic applications, or alternatively, inthe development of new therapeutic applications. For example, in sometherapeutic applications, e.g., in anti-neoplastic or acute caresettings where rapid drug clearance or turn-off may be desired, it isdesirable to decrease the residence times of aptamers in thecirculation. Alternatively, in other therapeutic applications, e.g.,maintenance therapies where systemic circulation of a therapeutic isdesired, it may be desirable to increase the residence times of aptamersin circulation.

[0229] In addition, the tunability of aptamer pharmacokinetics is usedto modify the biodistribution of an aptamer therapeutic in a subject.For example, in some therapeutic applications, it may be desirable toalter the biodistribution of an aptamer therapeutic in an effort totarget a particular type of tissue or a specific organ (or set oforgans). In these applications, the aptamer therapeutic preferentiallyaccumulates in a specific tissue or organ(s). In other therapeuticapplications, it may be desirable to target tissues displaying acellular marker or a symptom associated with a given disease, cellularinjury or other abnormal pathology, such that the aptamer therapeuticpreferentially accumulates in the affected tissue. For example, asdescribed herein, PEGylation of an aptamer therapeutic (e.g. PEGylationwith a 20 kDa PEG polymer) is used to target inflamed tissues, such thatthe PEGylated aptamer therapeutic preferentially accumulates in inflamedtissue.

[0230] The pharmacokinetic and biodistribution profiles of aptamertherapeutics (e.g., aptamer conjugates or aptamers having alteredchemistries, such as modified nucleotides) are determined by monitoringa variety of parameters. Such parameters include, for example, thehalf-life (t_(1/2)), the plasma clearance (C1), the volume ofdistribution (Vss), the area under the concentration-time curve (AUC),maximum observed serum or plasma concentration (C_(max)), and the meanresidence time (MRT) of an aptamer composition. As used herein, the term“AUC” refers to the area under the plot of the plasma concentration ofan aptamer therapeutic versus the time after aptamer administration. TheAUC value is used to estimate the bioavailability (i.e., the percentageof administered aptamer therapeutic in the circulation after aptameradministration) and/or total clearance (C1) (i.e., the rate at which theaptamer therapeutic is removed from circulation) of a given aptamertherapeutic. The volume of distribution relates the plasma concentrationof an aptamer therapeutic to the amount of aptamer present in the body.The larger the Vss, the more an aptamer is found outside of the plasma(i.e., the more extravasation).

[0231] The pharmacokinetic and biodistribution properties ofphosphorothioate-containing antisense oligonucleotides, which clearrapidly from circulation, and distribute into tissues (where eliminationoccurs slowly, as a result of metabolic degradation) are described inthe art: (See e.g., Srinivasan and Iversen (1995), J. Clin. Lab. Anal.9(2): 129-37; Agrawal and Zhang (1997), Ciba Found. Symp. 209: 60-75,discussion 75-8; Akhtar and Agrawal (1997), Trends Pharmacol. Sci.18(1): 12-8; Crooke (1997), Adv. Pharmacol. 40: 1-49; Grindel, et al.(1998), Antisense Nucleic Acid Drug Dev. 8(1): 43-52; Monteith and Levin(1999), Toxicol. Pathol. 27(1): 8-13; Peng, et al. (2001), AntisenseNucleic Acid Drug Dev. 11(1): 15-27). Early studies involving antisenseoligonucleotides have explored the effects of various conjugationchemistries on pharmacokinetics and biodistribution, with the ultimategoal of increasing delivery of antisense molecules to their sites ofaction inside cells or within certain tissue types in vivo (Antopolsky,et al. (1999), Bioconjug. Chem. 10(4): 598-606; Zubin, et al. (1999),FEBS Lett. 456(1): 59-62; Astriab-Fisher, et al. (2000), Biochem.Pharmacol. 60(1): 83-90; Lebedeva, et al., (2000), Eur. J. Pharm.Biopharm. 50(1): 101-19; Manoharan (2002), Antisense Nucleic Acid DrugDev. 12(2): 103-28). For example, conjugation with cholesterol has beenreported to increase the circulation half-life of antisenseoligonucleotides, most likely through association with plasmalipoproteins, and promoting hepatic uptake (de Smidt, et al. (1991),Nucleic Acids Res. 19(17): 4695-4700). Early work involving antisenseoligonucleotides has indicated that nonspecific protein-bindinginteractions play an important role in the rapid loss ofphosphorothioate-containing antisense oligonucleotide from circulationand distribution to tissues (See e.g., Srinivasan and Iversen (1995), J.Clin. Lab. Anal. 9(2): 129-37; Agrawal and Zhang (1997), Ciba Found.Symp. 209: 60-75, discussion 75-8; Akhtar and Agrawal (1997), TrendsPharmacol. Sci 18(1): 12-8; Crooke (1997), Adv. Pharmacol. 40: 1-49;Grindel, et al. (1998), Antisense Nucleic Acid Drug Dev. 8(1): 43-52;Monteith and Levin (1999), Toxicol. Pathol. 27(1): 8-13; Peng, et al.(2001), Antisense Nucleic Acid Drug Dev. 11(1): 15-27).

[0232] In contrast to antisense oligonucleotides, aptamers are generallylonger (30-40 vs. 10-20 nucleotides), possess different types ofchemical modifications (sugar modifications, e.g., 2′-F, 2′-O-Me,2′-NH₂, vs. backbone modifications), and assume complex tertiarystructures that are more resistant to degradation. Aptamers are, in manyrespects, more structurally similar to the three dimensional forms ofglobular proteins than to nucleic acids. Given these considerabledifferences, the in vivo disposition of aptamers is not readilypredictable from antisense results.

[0233] More recently, delivery peptides for carrying large polarmacromolecules, including oligonucleotides, across cellular membraneshave also been explored as a means to augment in vivo the range forapplication of antisense and other therapeutics. Examples of theseconjugates include a 13-amino acid fragment (Tat) of the HIV Tat protein(Vives, et al. (1997), J. Biol. Chem. 272(25): 16010-7), a 16-amino acidsequence derived from the third helix of the Drosophila antennapedia(Ant) homeotic protein (Pietersz, et al. (2001), Vaccine 19(11-12):1397-405), and short, positively charged cell-permeating peptidescomposed of polyarginine (Arg₇) (Rothbard, et al. (2000), Nat. Med.6(11): 1253-7; Rothbard, J et al. (2002), J. Med. Chem. 45(17): 3612-8).For example, the TAT peptide is described in U.S. Pat. Nos. 5,804,604and 5,674,980.

[0234] The present invention provides materials and methods to modulate,in a controlled manner, the pharmacokinetics and biodistribution ofstabilized aptamer compositions in vivo by conjugating an aptamer to amodulating moiety such as a small molecule, peptide, or polymer terminalgroup, or by incorporating modified nucleotides into an aptamer.Pharmacokinetics and biodistribution of aptamer conjugates in biologicalsamples are quantified radiometrically and by a hybridization-based dualprobe capture assay with enzyme-linked fluorescent readout. As describedherein, conjugation of a modifying moiety and/or altering nucleotide(s)chemical composition alter fundamental aspects of aptamer residence timein circulation and distribution to tissues.

[0235] Aptamers are conjugated to a variety of modifying moieties, suchas, for example, high molecular weight polymers, e.g., PEG, peptides,e.g., Tat, Ant and Arg₇, and small molecules, e.g., lipophilic compoundssuch as cholesterol. As shown herein, a mixed composition aptamercontaining both 2′F and 2′-OMe stabilizing modifications persistedsignificantly longer in the blood stream than did a fully2′-O-methylated composition. Among the conjugates prepared according tothe materials and methods of the present invention, in vivo propertiesof aptamers were altered most profoundly by complexation with PEGgroups. For example complexation of the mixed 2′F and 2′-OMe modifiedaptamer therapeutic with a 20 kDa PEG polymer hindered renal filtrationand promoted aptamer distribution to both healthy and inflamed tissues.Furthermore, the 20 kDa PEG polymer-aptamer conjugate proved nearly aseffective as a 40 kDa PEG polymer in preventing renal filtration ofaptamers. While one effect of PEGylation was on aptamer clearance, theprolonged systemic exposure afforded by presence of the 20 kDa moietyalso facilitated distribution of aptamer to tissues, particularly thoseof highly perfused organs and those at the site of inflammation. Theaptamer-20 kDa PEG polymer conjugate (ARC120) directed aptamerdistribution to the site of inflammation, such that the PEGylatedaptamer preferentially accumulated in inflamed tissue. In someinstances, the 20 kDa PEGylated aptamer conjugate was able to access theinterior of cells, such as, for example, kidney cells.

[0236] Overall, effects on aptamer pharmacokinetics and tissuedistribution produced by low molecular weight modifying moieties,including cholesterol and cell-permeating peptides were less pronouncedthan those produced as a result of PEGylation or modification ofnucleotides (e.g., an altered chemical composition). An aptamerconjugated to cholesterol showed more rapid plasma clearance relative tounconjugated aptamer, and a large volume of distribution, which suggestssome degree of aptamer extravasation. This result appears to contrastpublished data demonstrating the capacity of a cholesterol tag tosignificantly prolong the plasma half-life of an antisenseoligonucleotide (de Smidt et al., (1991), Nucleic Acids Res. 19(17):4695-4700). While not intending to be bound by theory, the resultsprovided herein, may suggest that cholesterol-mediated associations withplasma lipoproteins, postulated to occur in the case of the antisenseconjugate, are precluded in the particular context of theaptamer-cholesterol conjugate folded structure, and/or relate to aspectof the lipophilic nature of the cholesterol group. Like cholesterol, thepresence of a Tat peptide tag promoted clearance of aptamer from theblood stream, with comparatively high levels of conjugate appearing inthe kidneys at 48 hrs. Other peptides (e.g., Ant, Arg₇) that have beenreported in the art to mediate passage of macromolecules across cellularmembranes in vitro, did not appear to promote aptamer clearance fromcirculation. However, like Tat, the Ant conjugate significantlyaccumulated in the kidneys relative to other aptamers. While notintending to be bound by theory, it is possible that unfavorablepresentation of the Ant and Arg7 peptide modifying moieties in thecontext of three dimensionally folded aptamers in vivo impaired theability of these peptides to influence aptamer transport properties.

[0237] Prior to the invention described herein, little was knownconcerning the pharmacokinetics and biodistribution of oligonucleotideswith a 2′-OMe chemical composition (Tavitian, et al. (1998), Nat. Med.4(4): 467-71). For several reasons, incorporation of 2′-OMesubstitutions is a particularly attractive means to stabilize aptamersagainst nuclease attack. One attribute is safety: 2′-O-methylation isknown as a naturally occurring and abundant chemical modification ineukaryotic ribosomal and cellular RNAs. Human rRNAs are estimated tocontain roughly one hundred 2′-O-methylated sugars per ribosome (Smithand Steitz (1997), Cell 89(5): 669-72). Thus, aptamer compositionsincorporating 2′-OMe substitutions are expected to be non-toxic. Insupport of this view, in vitro and in vivo studies indicate that 2′-OMenucleotides are not readily polymerized by human DNA polymerases (α orγ), or by human DNA primase, and thus, pose a low risk for incorporationinto genomic DNA (Richardson, et al. (2000), Biochem. Pharmacol. 59(9):1045-52; Richardson, et al. (2002), Chem. Res. Toxicol. 15(7): 922-6).Additionally, from a cost of goods perspective, pricing per gram forsynthesis of 2′-OMe containing oligonucleotides is less than the pricingper gram for both 2′-F and 2′-OH containing RNAs.

[0238] A comparison of a mixed 2′F/2′-OMe composition aptamer andconjugated aptamers was conducted in vivo to determine plasma clearance.The unconjugated test aptamer which incorporates both 2′-F and 2′-OMestabilizing chemistries, is typical of current generation aptamers as itexhibits a high degree of nuclease stability in vitro and in vivo.Compared to the mixed 2′F/2′-OMe composition aptamer, unmodified aptamerdisplayed rapid loss from plasma (i.e., rapid plasma clearance) and arapid distribution into tissues, primarily into the kidney.

[0239] Tests can be conducted to determine whether the hydrophobicnature of a fully 2′-OMe modified aptamer renders the oligonucleotidemore prone to nonspecific associations with plasma or cellular component(as is the case with antisense oligonucleotides). In addition,experiments can be conducted to define the protein-binding properties of2′-OMe-modified aptamers. While not intending to be bound by theory,levels of full-length all-2′-O-methyl substituted aptamer abovebackground were detected in several tissues, kidney, liver, and spleen,even at 48 hrs after dosing, possibly due to the extreme robustness ofthe fully 2′-OMe aptamer towards nuclease digestion. In one example,consistent with its plasma clearance profile and distribution to thekidney, a fully 2′-OMe aptamer was eliminated rapidly via the urine.

[0240] When expressed as percent of administered dose, all aptamers orconjugates examined herein showed significant levels of distribution tokidney, liver, and gastrointestinal tract. When corrected fororgan/tissue weight, highest mass-normalized concentrations of aptamerswere seen in highly perfused organs (kidneys, liver, spleen, heart,lungs) and unexpectedly, mediastinal lymph nodes. Since aptamers arebioavailable (up to 80%) following subcutaneous injection (Tucker etal., (1999), J. Chromatography B. 732: 203-212), they are expected tohave access to targets in the lymphatic system through this route ofadministration. Ready access to the lymphatics via intravenous dosing isof interest from the standpoint of developing aptamer therapeutics forinfectious disease indications such as HIV/AIDS. Thus, aptamertherapeutics conjugated to modifying moieties and aptamers havingaltered chemistries (e.g., including modified nucleotides) will beuseful in the treatment of infectious diseases such as HIV/AIDS.

[0241] Consistent with its enhanced plasma pharmacokinetics, theconcentration of 20 kDa PEGylated aptamer detected in highly perfusedorgans was higher than for the other aptamers that were assayed. As ageneral trend, aptamer concentrations measured in the kidneys decreasedwith time, with exception of 20 kDa PEGylated aptamer, whereconcentrations remained roughly constant over time. Conversely, in liverconcentrations of all aptamers remained roughly constant, except for 20kDa PEGylated aptamer, whose levels decreased with time. Thesedifferences may be understood in terms of the extended plasma half-lifeof the 20 kDa PEG conjugate and its increased uptake in highly perfusedorgans. While one of the effects of complexation with a 20 kDa PEGmodifying moiety was to retard renal filtration of the aptamerconjugate, the comparatively high concentrations of the 20 kDa PEGconjugate measured in well-perfused organs, relative to other aptamersor conjugates, suggested that PEGylation can modulate aptamerdistribution to tissues, as well as promote extended plasma half-life(t_(1/2)). As described herein, the 20 kDa PEGylated aptamer-conjugatemodulated aptamer distribution to tissues. The level of the 20 kDaPEGylated aptamer detected in inflamed tissues was higher than for theother aptamers that were assayed, and, in some instances, the aptamerwas able to access the interior of cells (e.g. kidney cells).

[0242] While not intending to be bound by theory, it is speculated thatprolonged residence in the blood stream increases exposure of conjugatedaptamer to tissues, leading to enhanced uptake, which is most pronouncedin the case of highly perfused organs and in the case of inflamedtissues. The presence of aptamer in residual blood may contribute to,but is unlikely to account entirely for, the increased levels of the 20kDa aptamer conjugate in perfused organs and inflamed tissue shownherein. The enhanced distribution of PEGylated aptamer to perfusedorgans and inflamed tissues represents extravasation, as suggested byexperiments in mice dosed with tritiated 20 kDa PEG conjugate where [³H]signal was seen in cells of both the liver and kidney (See Examplesprovided below). Early work on aptamer therapeutics focused primarily ondevelopment of aptamers complexed with higher molecular weight (40 kDa)PEG species in an effort to avoid renal filtration (Reyderman andStavchansky (1998), Pharmaceutical Research 15(6): 904-10; Tucker etal., (1999), J. Chromatography B. 732: 203-212; Watson, et al. (2000),Antisense Nucleic Acid Drug Dev. 10(2): 63-75; Carrasquillo, et al.(2003), Invest. Ophthalmology Vis. Sci. 44(1): 290-9). The presentinvention indicates that complexation with a smaller, e.g., 20 kDa, PEGpolymer sufficiently protects aptamer-based drugs from renal filtrationfor many therapeutic indications. Smaller PEGs (e.g., 10 kDa to 20 kDaPEG moieties) also provide the collateral benefits of ease of synthesisand reduced cost of goods, as compared to larger PEGs.

[0243] PEG-Derivatized Nucleic Acids

[0244] Derivatization of nucleic acids with high molecular weightnon-immunogenic polymers has the potential to alter the pharmacokineticand pharmacodynamic properties of nucleic acids making them moreeffective therapeutic agents. Favorable changes in activity can includeincreased resistance to degradation by nucleases, decreased filtrationthrough the kidneys, decreased exposure to the immune system, andaltered distribution of the therapeutic through the body.

[0245] The aptamer compositions of the invention may be derivatized withpolyalkylene glycol (PAG) moieties. Examples of PAG-derivatized nucleicacids are found in U.S. patent application Ser. No. 10/718,833, filed onNov. 21, 2003, which is herein incorporated by reference in itsentirety. Typical polymers used in the invention include poly(ethyleneglycol) (PEG), also known as or poly(ethylene oxide) (PEO) andpolypropylene glycol (including poly isopropylene glycol). Additionally,random or block copolymers of different alkylene oxides (e.g., ethyleneoxide and propylene oxide) can be used in many applications. In its mostcommon form, a polyalkylene glycol, such as PEG, is a linear polymerterminated at each end with hydroxyl groups:HO—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OH. This polymer, alpha-,omega-dihydroxylpoly(ethylene glycol), can also be represented asHO—PEG-OH, where it is understood that the —PEG-symbol represents thefollowing structural unit: —CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂— where ntypically ranges from about 4 to about 10,000.

[0246] As shown, the PEG molecule is di-functional and is sometimesreferred to as “PEG diol.” The terminal portions of the PEG molecule arerelatively non-reactive hydroxyl moieties, the —OH groups, that can beactivated, or converted to functional moieties, for attachment of thePEG to other compounds at reactive sites on the compound. Such activatedPEG diols are referred to herein as bi-activated PEGs. For example, theterminal moieties of PEG diol have been functionalized as activecarbonate ester for selective reaction with amino moieties bysubstitution of the relatively nonreactive hydroxyl moieties, —OH, withsuccinimidyl active ester moieties from N-hydroxy succinimide.

[0247] In many applications, it is desirable to cap the PEG molecule onone end with an essentially non-reactive moiety so that the PEG moleculeis mono-functional (or mono-activated). In the case of proteintherapeutics which generally display multiple reaction sites foractivated PEGs, bi-functional activated PEGs lead to extensivecross-linking, yielding poorly functional aggregates. To generatemono-activated PEGs, one hydroxyl moiety on the terminus of the PEG diolmolecule typically is substituted with non-reactive methoxy end moiety,—OCH₃. The other, un-capped terminus of the PEG molecule typically isconverted to a reactive end moiety that can be activated for attachmentat a reactive site on a surface or a molecule such as a protein.

[0248] PAGs are polymers which typically have the properties ofsolubility in water and in many organic solvents, lack of toxicity, andlack of immunogenicity. One use of PAGs is to covalently attach thepolymer to insoluble molecules to make the resulting PAG-molecule“conjugate” soluble. For example, it has been shown that thewater-insoluble drug paclitaxel, when coupled to PEG, becomeswater-soluble. Greenwald, et al., J. Org. Chem., 60:331-336 (1995). PAGconjugates are often used not only to enhance solubility and stabilitybut also to prolong the blood circulation half-life of molecules.

[0249] Polyalkylated compounds of the invention are typically between 5and 80 kD in size. Other PAG compounds of the invention are between 10and 80 kD in size. Still other PAG compounds of the invention arebetween 10 and 60 kD in size. For example, a PAG polymer may be at least10, 20, 30, 40, 50, 60, or 80 kD in size. Such polymers can be linear orbranched.

[0250] In contrast to biologically-expressed protein therapeutics,nucleic acid therapeutics are typically chemically synthesized fromactivated monomer nucleotides. PEG-nucleic acid conjugates may beprepared by incorporating the PEG using the same iterative monomersynthesis. For example, PEGs activated by conversion to aphosphoramidite form can be incorporated into solid-phaseoligonucleotide synthesis. Alternatively, oligonucleotide synthesis canbe completed with site-specific incorporation of a reactive PEGattachment site. Most commonly this has been accomplished by addition ofa free primary amine at the 5′-terminus (incorporated using a modifierphosphoramidite in the last coupling step of solid phase synthesis).Using this approach, a reactive PEG (e.g., one which is activated sothat it will react and form a bond with an amine) is combined with thepurified oligonucleotide and the coupling reaction is carried out insolution.

[0251] The ability of PEG conjugation to alter the biodistribution of atherapeutic is related to a number of factors including the apparentsize (e.g., as measured in terms of hydrodynamic radius) of theconjugate. Larger conjugates (>10 kDa) are known to more effectivelyblock filtration via the kidney and to consequently increase the serumhalf-life of small macromolecules (e.g., peptides, antisenseoligonucleotides). The ability of PEG conjugates to block filtration hasbeen shown to increase with PEG size up to approximately 50 kDa (flirterincreases have minimal beneficial effect as half life becomes defined bymacrophage-mediated metabolism rather than elimination via the kidneys).

[0252] Production of high molecular weight PEGs (>10 kDa) can bedifficult, inefficient, and expensive. As a route towards the synthesisof high molecular weight PEG-nucleic acid conjugates, previous work hasbeen focused towards the generation of higher molecular weight activatedPEGs. One method for generating such molecules involves the formation ofa branched activated PEG in which two or more PEGs are attached to acentral core carrying the activated group. The terminal portions ofthese higher molecular weight PEG molecules, i.e., the relativelynon-reactive hydroxyl (—OH) moieties, can be activated, or converted tofunctional moieties, for attachment of one or more of the PEGs to othercompounds at reactive sites on the compound. Branched activated PEGswill have more than two termini, and in cases where two or more terminihave been activated, such activated higher molecular weight PEGmolecules are referred to herein as, multi-activated PEGs. In somecases, not all termini in a branch PEG molecule are activated. In caseswhere any two termini of a branch PEG molecule are activated, such PEGmolecules are referred to as bi-activated PEGs. In some cases where onlyone terminus in a branch PEG molecule is activated, such PEG moleculesare referred to as mono-activated. As an example of this approach,activated PEG prepared by the attachment of two monomethoxy PEGs to alysine core which is subsequently activated for reaction has beendescribed (Harris et al., Nature, vol.2: 214-221, 2003).

[0253] The present invention provides another cost effective route tothe synthesis of high molecular weight PEG-nucleic acid (preferably,aptamer) conjugates including multiply PEGylated nucleic acids. Thepresent invention also encompasses PEG-linked multimericoligonucleotides, e.g., dimerized aptamers. The present invention alsorelates to high molecular weight compositions where a PEG stabilizingmoiety is a linker which separates different portions of an aptamer,e.g., the PEG is conjugated within a single aptamer sequence, such thatthe linear arrangement of the high molecular weight aptamer compositionis, e.g., nucleic acid—PEG—nucleic acid—PEG—nucleic acid.

[0254] High molecular weight compositions of the invention include thosehaving a molecular weight of at least 10 kD. Compositions typically havea molecular weight between 10 and 80 kD in size. High molecular weightcompositions of the invention are at least 10, 20, 30, 40, 50, 60, or 80kD in size.

[0255] A stabilizing moiety is a molecule, or portion of a molecule,which improves pharmacokinetic and pharmacodynamic properties of thehigh molecular weight aptamer compositions of the invention. In somecases, a stabilizing moiety is a molecule or portion of a molecule whichbrings two or more aptamers, or aptamer domains, into proximity, orprovides decreased overall rotational freedom of the high molecularweight aptamer compositions of the invention. A stabilizing moiety canbe a polyalkylene glycol, such a polyethylene glycol, which can belinear or branched, a homopolymer or a heteropolymer. Other stabilizingmoieties include polymers such as peptide nucleic acids (PNA).Oligonucleotides can also be stabilizing moieties; such oligonucleotidescan include modified nucleotides, and/or modified linkages, such asphosphorothioates. A stabilizing moiety can be an integral part of anaptamer composition, i.e., it is covalently bonded to the aptamer.

[0256] Compositions of the invention include high molecular weightaptamer compositions in which two or more nucleic acid moieties arecovalently conjugated to at least one polyalkylene glycol moiety. Thepolyalkylene glycol moieties serve as stabilizing moieties. Incompositions where a polyalkylene glycol moiety is covalently bound ateither end to an aptamer, such that the polyalkylene glycol joins thenucleic acid moieties together in one molecule, the polyalkylene glycolis said to be a linking moiety. In such compositions, the primarystructure of the covalent molecule includes the linear arrangementnucleic acid-PAG-nucleic acid. One example is a composition having theprimary structure nucleic acid-PEG-nucleic acid. Another example is alinear arrangement of: nucleic acid—PEG—nucleic acid—PEG—nucleic acid.

[0257] To produce the nucleic acid—PEG—nucleic acid conjugate, thenucleic acid is originally synthesized such that it bears a singlereactive site (e.g., it is mono-activated). In a preferred embodiment,this reactive site is an amino group introduced at the 5′-terminus byaddition of a modifier phosphoramidite as the last step in solid phasesynthesis of the oligonucleotide. Following deprotection andpurification of the modified oligonucleotide, it is reconstituted athigh concentration in a solution that minimizes spontaneous hydrolysisof the activated PEG. In a preferred embodiment, the concentration ofoligonucleotide is 1 mM and the reconstituted solution contains 200 mMNaHCO₃-buffer, pH 8.3. Synthesis of the conjugate is initiated by slow,step-wise addition of highly purified bi-functional PEG. In a preferredembodiment, the PEG diol is activated at both ends (bi-activated) byderivatization with succinimidyl propionate. Following reaction, thePEG-nucleic acid conjugate is purified by gel electrophoresis or liquidchromatography to separate fully-, partially-, and un-conjugatedspecies. Multiple PAG molecules concatenated (e.g., as random or blockcopolymers) or smaller PAG chains can be linked to achieve variouslengths (or molecular weights). Non-PAG linkers can be used between PAGchains of varying lengths.

[0258] The 2′-O-methyl, 2′-fluoro modifications stabilize the aptameragainst nucleases and increase its half life in vivo. The 3′-3′-dT capalso increases exonuclease resistance. See, e.g., U.S. Pat. Nos.5,674,685; 5,668,264; 6,207,816; and 6,229,002, each of which isincorporated by reference herein in its entirety.

[0259] PAG-Derivatization of a Reactive Nucleic Acid

[0260] High molecular weight PAG-nucleic acid-PAG conjugates can beprepared by reaction of a mono-functional activated PEG with a nucleicacid containing more than one reactive site. In one embodiment, thenucleic acid is bi-reactive, or bi-activated, and contains two reactivesites: a 5′-amino group and a 3′-amino group introduced into theoligonucleotide through conventional phosphoramidite synthesis, forexample: 3′-5′-di-PEGylation as illustrated in FIG. 22. In alternativeembodiments, reactive sites can be introduced at internal positions,using for example, the 5-position of pyrimidines, the 8-position ofpurines, or the 2′-position of ribose as sites for attachment of primaryamines. In such embodiments, the nucleic acid can have several activatedor reactive sites and is said to be multiply activated. Followingsynthesis and purification, the modified oligonucleotide is combinedwith the mono-activated PEG under conditions that promote selectivereaction with the oligonucleotide reactive sites while minimizingspontaneous hydrolysis. In the preferred embodiment, monomethoxy-PEG isactivated with succinimidyl propionate and the coupled reaction iscarried out at pH 8.3. To drive synthesis of the bi-substituted PEG,stoichiometric excess PEG is provided relative to the oligonucleotide.Following reaction, the PEG-nucleic acid conjugate is purified by gelelectrophoresis or liquid chromatography to separate fully-, partially-,and un-conjugated species.

[0261] The linking domains can also have one ore more polyalkyleneglycol moieties attached thereto. Such PAGs can be of varying lengthsand may be used in appropriate combinations to achieve the desiredmolecular weight of the composition.

[0262] The effect of a particular linker can be influenced by both itschemical composition and length. A linker that is too long, too short,or forms unfavorable steric and/or ionic interactions with the targetwill preclude the formation of complex between aptamer and target. Alinker, which is longer than necessary to span the distance betweennucleic acids, may reduce binding stability by diminishing the effectiveconcentration of the ligand. Thus, it is often necessary to optimizelinker compositions and lengths in order to maximize the affinity of anaptamer to a target.

[0263] All publications and patent documents cited herein areincorporated herein by reference as if each such publication or documentwas specifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

EXAMPLES Example 1 Large Scale Aptamer Synthesis and Conjugation

[0264] ARC126 (5′-(SEQ ID NO:1)-HEG-(SEQ ID NO:2)-HEG-(SEQ ID NO:3)-3′dT-3′) is a 29 nucleotide aptamer (excluding an inverted T at the 3′end) specific for PDGF which contains a 3′ inverted-dT cap for enhancedstability against nuclease attack. PEG moieties can be conjugated toARC126 by using a 5′-amino terminus modifier for subsequent conjugationreactions. Syntheses were performed using standard solid-phasephosphoramidite chemistry. The oligonucleotide was deprotected withammonium hydroxide/methylamine (1:1) at room temperature for 12 hoursand purified by ion exchange HPLC. ARC 128 (5′-(SEQ ID No.4)-HEG-(SEQ IDNO:5)-HEG-(SEQ ID NO:6)-3′), an inactive variant which no longer bindsPDGF, was synthesized using the same method.

[0265] ARC126 was conjugated to several different PEG moieties: 20 kDaPEG (ARC240, (5′-[20K PEG]-(SEQ ID NO:1)-HEG-(SEQ ID NO:2)-HEG-(SEQ IDNO:3)-3′ dT-3′)); 30 kDa PEG (ARC308, (5′-[30K PEG]-(SEQ IDNO:1)-HEG-(SEQ ID NO:2)-HEG-(SEQ ID NO:3)-3′ dT-3′)); 40 kDa PEG(ARC127, (5′-[40K PEG]-(SEQ ID NO:1)-HEG-(SEQ ID NO:2)-HEG-(SEQ IDNO:3)-3′ dT-3′)). ARC126 was dissolved to 2 mM in 100 mM sodiumcarbonate buffer, pH 8.5, and was reacted for 1 hour with a 2.5 molarexcess of mPEG-SPA (MW 20 kDa) or mPEG2-NHS ester (MW 40 kDa)(Shearwater Corp., Huntsville, Ala.), and 3.5 molar excess (for 24hours) of mPEG-nPNC (MW 30 kDa) (NOF Corporation, Tokyo, Japan) in equalvolumes of acetonitrile. The resulting products were then purified byion exchange HPLC on a 50 ml Super Q 5PW column (Tosoh Bioscience,Montgomeryville, Pa.) using aqueous NaCl as eluent. The conjugatedoligonucleotides were then desalted on a 100 ml Amberchrom CG300S(Tosoh) column using a water/acetonitrile gradient. Aptamer conjugateswere lyophilized for storage.

[0266] In order to be able to use ARC127 in animal models, the qualityof the material synthesized needed to be tested for endotoxin levels.Endotoxin content of synthesized ARC127 was determined using the LALtest (performed by Nelson Labs, AZ). Results for endotoxin testing areshown in Table 1 below. The detected quantities of endotoxin were belowthe ISO standard for sterile irrigation solutions (0.5 EU/mL), i.e.lower than levels allowed for IV administration. This indicated that ARC126 and ARC127 preparations were sterile and that it was possible toproceed to animal efficacy models. TABLE 1 Endotoxin levels in largescale synthesis of therapeutic aptamers. Spike Sample Dilution Endotoxindetected recovery ARC126 1:10  2.5 EU/ml&0.52 EU/mg 73% 1:100  2.8EU/ml&.58 EU/mg 103% 1:200  2.5 EU/ml&0.52 EU/mg 104% ARC127 1:1 0.19EU/ml&0.17 EU/mg 51% 1:10 0.33 EU/ml&0.30 EU/mg 97% 1:100 0.45EU/ml&0.41 EU/mg 115% ARC128 1:1 0.52 EU/ml&0.76 EU/mg 105% 1:10 0.57EU/ml&0.82 EU/mg 127% 1:100 0.43 EU/ml&0.62 EU/mg 136%

[0267] Small scale syntheses of the de-fluorinated ARC-126 aptamervariants were done on Applied Biosystems' Expedite 8909 DNA (FosterCity, Calif.) synthesizer using standard solid-phase phosphoramiditechemistry and vendor's recommended coupling protocols. The aptamers werecleaved and deprotected by adding 250 μL ammonium hydroxide/40% aqueousmethylamine (1:1) to column support and placed in a 65° C. heating blockfor 30 minutes. Aptamers were dried down in a Speed Vac (Savant), thenresuspended in 200 μL diH₂O. HPLC purification was performed on aTransgenomic WAVE HPLC (Omaha, Nebr.). The columns used for ion-exchangeare the DNAPAC (Dionex, Sunnyvale, Calif.) and Resource (AmershamBiosciences, Piscataway, N.J.). Buffer A: 25 mM sodium phosphate/25%acetonitrile; Buffer B: 25 mM sodium phosphate/400 mM sodiumperchlorate/25% acetonitrile, a gradient from 0-80% B was used. Purifiedfractions were then pooled and dried down in a Speed Vac and resuspendedin 200 μL diH₂O.

Example 2 Stability Studies with Fluorinated Aptamers

[0268] ARC126 and ARC127 freshly synthesized in house were compared toARC126 and ARC127 that were synthesized by Proligo (Boulder, Colo.), andhad been stored lyophilized for 2 years at −20° C. (legacy aptamers).

[0269] ARC127 synthesized in house and legacy ARC 127 were passed overan ion-exchange HPLC column for analysis. FIG. 5A is a trace of anion-exchange HPLC analysis of freshly synthesized and legacy ARC 127showing that after 2 years storage lyophilized at −20° C., relatively nodegradation of the legacy ARC127 was detected.

[0270] The legacy ARC 126 and ARC127 aptamers stored at −20° C. for twoyears were also tested for potency, and compared to freshly synthesizedARC126 and ARC127 synthesized in house using the 3T3 cell proliferationassay (Example 3). FIG. 5B shows cell-based assay results for potencydemonstrating that even after lyophilization and storage at −20 degreesfor 2 years, the legacy aptamers were just as potent as ARC126 andARC127 newly synthesized in house.

Example 3 Composition and Sequence Optimization of ARC126 Variants

[0271] The sequence and secondary structure of the anti-PDGF aptamerdesignated ARC126 is shown in FIG. 6A. The sequence and secondarystructure of derivatives of ARC 126 in which the nucleotides with2′-fluoro-substituents have been replaced are shown in FIG. 6B. Theloops shown at the termini of the two internal stems are polyethyleneglycol-6 (PEG-6) spacers and modified nucleotides are represented bydA=deoxyadenosine; dG=deoxyguanosine; mA=2′-O-methyladenosine;dT=deoxythymidine; dC=deoxycytosine; mT=2′-O-methylthymidine;mG=2′-O-methylguanosine; mC=2′-O-methylcytosine; [3′T]=inverteddeoxythymidine; fC=2′-fluorocytosine; and fU=2′-fluorouridine.

[0272] As shown in FIG. 6A, the 29-nucleotide composition of ARC126contains seven 2′-fluoro residues (three 2′-fluorouridines and four2′-fluorocytosines). Due to considerations including genotoxicity ofbreakdown products, the compositional optimization of ARC126 with thegoal of modifying the sequence composition to remove all or most of2′-fluoro residues without compromising the potency or stability of theexisting molecule. One avenue for removal of 2′-fluoro residuesconsisted of simple substitution of all seven 2′-fluoro residues fordeoxy residues. Such an all-DNA variant of ARC126, designated ARC299(SEQ ID NO:59-PEG-SEQ ID NO:60-PEG-SEQ ID NO:61, wherein PEG=PEG-6spacer), was synthesized and tested in in vitro biochemical binding andcell-based proliferation assays. These experiments showed that simplesubstitution of all 2′-fluoro residues for deoxy residues in the 29-mercomposition of ARC126 induced instability in the central and upperstems, leading to significantly reduced activity/potency.

[0273] The second approach taken to effect removal of 2′-fluoro residuesfrom ARC126 was the substitution, either singly or in blocks, of2′-O-methyl residues for 2′-fluoro residues to ameliorate the relativebase-pairing instability in the central and upper stems observed withthe all-deoxy composition. A number of composition variants weresynthesized, representing single point-substitutions of 2′-O-methyl ordeoxy residues for 2′-fluoro residues (ARC277, 5′-(SEQ IDNO:56)-PEG-(SEQ ID NO:57)-PEG-(SEQ ID NO:58)-3T-3′), as well as blocksubstitutions of 2′-O-methyl residues for 2′-fluoro residues (ARC337,5′-(SEQ ID NO:59)-PEG-(SEQ ID NO:60)-PEG-(SEQ ID NO:69)-3T-3′; ARC338,5′-(SEQ ID NO:70)-PEG-(SEQ ID NO:60)-PEG-(SEQ ID NO:71)-3T-3′; ARC339,5′-(SEQ ID NO:65)-PEG-(SEQ ID NO:60)-PEG-(SEQ ID NO:72)-3T-3′; ARC340,5′-(SEQ ID NO:67)-PEG-(SEQ ID NO:60)-PEG-(SEQ ID NO:69)-3T-3′;combinations of single and block substitutions (ARC341, 5′-(SEQ IDNO:99)-PEG-(SEQ ID NO:54)-PEG-(SEQ ID NO:58)-3T-3′; ARC342, 5′-(SEQ IDNO:73)-PEG-(SEQ ID NO:54)-PEG-(SEQ ID NO:58)-3T-3′; ARC344, 5′-(SEQ IDNO:74)-PEG-(SEQ ID NO:54)-PEG-(SEQ ID NO:58)-3T-3′; ARC345, 5′-(SEQ IDNO:99)-PEG-(SEQ ID NO:75)-PEG-(SEQ ID NO:58)-3T-3′; ARC346, 5′-(SEQ IDNO:99)-PEG-(SEQ ID NO:76)-PEG-(SEQ ID NO:58)-3T-3′; ARC347, 5′-(SEQ IDNO:77)-PEG-(SEQ ID NO:78)-PEG-(SEQ ID NO:79)-3T-3′; ARC362, 5′-(SEQ IDNO:99)-PEG-(SEQ ID NO:54)-PEG-(SEQ ID NO:80)-3T-3′; ARC363, 5′-(SEQ IDNO:81)-PEG-(SEQ ID NO:54)-PEG-(SEQ ID NO:82)-3T-3′; ARC364, 5′-(SEQ IDNO:67)-PEG-(SEQ ID NO:60)-PEG-(SEQ ID NO:83)-3T-3′; ARC365, 5′-(SEQ IDNO:56)-PEG-(SEQ ID NO:57)-PEG-(SEQ ID NO:83)-3T-3′; ARC366, 5′-(SEQ IDNO:84)-PEG-(SEQ ID NO:57)-PEG-(SEQ ID NO:83)-3T-3′; ARC404, 5′-(SEQ IDNO:73)-PEG-(SEQ ID NO:57)-PEG-(SEQ ID NO:58)-3T-3′; ARC405, 5′-(SEQ IDNO:74)-PEG-(SEQ ID NO:57)-PEG-(SEQ ID NO:58)-3T-3′; ARC406, 5′-(SEQ IDNO:56)-PEG-(SEQ ID NO:75)-PEG-(SEQ ID NO:58)-3T-3′; ARC407, 5′-(SEQ IDNO:56)-PEG-(SEQ ID NO:76)-PEG-(SEQ ID NO:58)-3T-3′; ARC408, 5′-(SEQ IDNO:56)-PEG-(SEQ ID NO:57)-PEG-(SEQ ID NO:80)-3T-3′; ARC409, 5′-(SEQ IDNO:56)-PEG-(SEQ ID NO:57)-PEG-(SEQ ID NO:81)-3T-3′; ARC410, 5′-(SEQ IDNO:56)-PEG-(SEQ ID NO:57)-PEG-(SEQ ID NO:69)-3T-3′; ARC513 (5′-(SEQ IDNO:86)-PEG-(SEQ ID NO:57)-PEG-(SEQ ID NO:87) -3′ dT-3′), ARC514 (5′-(SEQID NO:86)-PEG-(SEQ ID NO:57)-PEG-(SEQ ID NO:88)-3′ dT-3′), ARC515(5′-(SEQ ID NO:86)-PEG-(SEQ ID NO:57)-PEG-(SEQ ID NO:89)-3′ dT-3′), andARC516 (5′-(SEQ ID NO:86)-PEG-(SEQ ID NO:57)-PEG-(SEQ ID NO:90)-3′dT-3′), and finally an all-2′-O-methyl composition (ARC300 (or 300B),5′-(SEQ ID NO:62)-PEG-(SEQ ID NO:63)-PEG-(SEQ ID NO:64)-3T-3′). Othercomposition variants include: ARC276, 5′-(SEQ ID NO:99)-PEG-(SEQ IDNO:54)-PEG-(SEQ ID NO:55)-3T-3′; ARC335, 5′-(SEQ ID NO:65)-PEG-(SEQ IDNO:60)-PEG-(SEQ ID NO:66)-3T-3′; ARC336, 5′-(SEQ ID NO:67)-PEG-(SEQ IDNO:60)-PEG-(SEQ ID NO:68)-3T-3′; ARC343, 5′-(SEQ ID NO:70)-PEG-(SEQ IDNO:60)-PEG-(SEQ ID NO:58)-3T-3′.

[0274] Table 2 below summarizes the sequence and composition of allARC126 variants synthesized and tested. Sequences shown are listed 5′→3′from left to right in the table. Table 2 also summarizes the compositionidentity and mean affinity and activity of all ARC126 variantssynthesized and tested in in vitro assays (competitive binding assay and3T3 proliferation assay) described below in Example 5. In the table,d=deoxy residue; f=2′-fluoro residue; m=2′-O-methyl residue;PEG=polyethylene glycol (PEG-6) spacer; 3T=inverted deoxythymidine.TABLE 2 Sequence and composition of ARC126 variants. SEQ ID 5′ Acti- No.ARC# modif. Sequence (5′ ≧ 3′) vity Affinity Activity 127 PEG40K-      dC dA dG dG dC fU dA fC mG -PEG- dC dG dT dA mG dA mG dC +++ 0.12320.66667       dA fU fC mA -PEG- dT dG dA dT fC fC fU mG -3T 124      dC dA dC dA dG dG dC dT dA dC    dG dG dC dA dC dG dT dA ++++ 0.213.45       dG dA dG dC dA    dT dC dA dC dC dA dT dG dA       dT dC dCdT dG dT dG -3T 276       dC dA dG dG dC dT dA dC mG -PEG- dC dG dT dAmG dA mG dC +++ 0.23 18       dA dT dC mA -PEG- dT dG dA dT dC dC dT mG-3T 277       dC dA dG dG dC mU dA mC mG -PEG- dC dG dT dA mG dA mG dC+++ 0.1 8.666667       dA mU mC mA -PEG- dT dG dA aT mC mC mU mG -3T 299      dC dA dG dG dC dT dA dC dG -PEG- dC dG dT dA dG dA dG dC − 2.11000       dA dT dC dA -PEG- dT dG dA dT dC dC dT dG -3T 300       mC mAmG mG mC mU mA mC mG -PEG- mC mG mU mA mG mA mG mC − 900       mA mU mCmA -PEG- mU mG mA mU mC mC mU mG -3T 335    dA dC dA dG dG dC dT dA dCdG -PEG- dC dG dT dA dG dA dG dC ++ 0.13 90    dA dT dC dA -PEG- dT dGdA dT dC dC dT dG dT -3T 336 dC dA dC dA dG dG dC dT dA dC dG -PEG- dCdG dT dA dG dA dG dC + 1.6 196.6667 dA dT dC dA -PEG- dT dG dA dT dC dCdT dG dT dG -3T 337       dC dA dG dG dC dT dA dC dG -PEG- dC dG dT dAdG dA dG dC − 1 1000       dA dT dC dA -PEG- dT dG dA dT mC mC mU mG -3T338       mC mA mG mG dC dT dA dC dG -PEG- dC dG dT dA dG dA dG dC − 151000       dA dT dC dA -PEG- dT dG dA dT dC dC dT dG -3T 339    dA dC dAdG dG dC dT dA dC dG -PEG- dC dG dT dA dG dA dG dC − 0.67 766.6667    dAdT dC dA -PEG- dT dG dA dT mC mC mU m mU -3T 340 dC dA dC dA dG dG dC dTdA dG dC -PEG- dC dG dT dA dG dA dG dC − 0.75 866.6667 dA dT dC dA -PEG-dT dG dA dT mC mC mU mG mU mG -3T 341       dC dA dG dG dC dT dA dC mG-PEG- dC dG dT dA mG dA mG dC − 24 1000       dA dT dC mA -PEG- dT dG dAdT mC mC mU mG -3T 343       mC mA mG mG dC dT dA dC dG -PEG- dC dG dTdA dG dA dG dC NA       dA dT dC dA -PEG- dT dG dA dT mC mC mU mG -3T342       dC dA dG dG dC dT dA mC mG -PEG- dC dG dT dA mG dA mG dC − 3.71000       dA dT dC mA -PEG- dT dG dA dT mC mC mU mG -3T 344       dC dAdG dG dC mU dA dC mG -PEG- dC dG dT dA mG dA mG dC + 1.7 200       dA dTdC mA -PEG- dT dG dA dT mC mC mU mG -3T 345       dC dA dG dG dC dT dAdC mG -PEG- dC dG dT dA mG dA mG dC +++ 0.52 13       dA dT mC mA -PEG-dT dG dA dT mC mC mU mG -3T 346       dC dA dG dG dC dT dA dC mG -PEG-dC dG dT dA mG dA mG dC +++ 0.37 15       dA mU dC mA -PEG- dT dG dA dTmC mC mU mG -3T 347       dC dA dG dG mC mU dA mC dG -PEG- mC dG mU dAdG dA dG mC − >1000 1000       dA mU mC dA -PEG- mU dG dA mU mC mC mU dG-3T 362       dC dA dG dG dC dT dA dC mG -PEG- dC dG dT dA mG dA mG dC +315       dA dT dC mA -PEG- dT dG dA dT dC mC mU mG -3T 363 dC dA dC dAdG dG dC dT dA dC mG -PEG- dC dG dT dA mG dA mG dC ++++ 3.923333 dA dTdC mA -PEG- dT dG dA dT dC dC dT dG dT dG -3T 364 dC dA dC dA dG dG dCdT dA dC dG -PEG- dC dG dT dA dG dA dG dC − 1719 dA dT dC dA -PEG- dT dGdA dT dC mC mU mG mU mG -3T 365 NH2—       dC dA dG dG dC mU dA mC mG-PEG- dC dG dT dA mG dA mG dC +++ 6.793333       dA mU mC mA -PEG- dT dGdA dT mC mC mU mG -3T 366 dC dA dC dA dG dG dC mU dA mC mG -PEG- dC dGdT dA mG dA mG dC +++ 5.535 dA mU mC mA -PEG- dT dG dA dT dC mC mU mG mUmG -3T 404       dC dA dG dG dC dT dA mC mG -PEG- dC dG dT dA mG dA mGdC ++++ 3.89       dA mU mC mA -PEG- dT dG dA dT mC mC mU mG -3T 405      dC dA dG dG dC mU dA dC mG -PEG- dC dG dT dA mG dA mG dC +++8.653333       dA mU mC mA -PEG- dT dG dA dT mC mC mU mG -3T 406      dC dA dG dG dC mU dA mC mG -PEG- dC dG dT dA mG dA mG dC +++ 17.15      dA dT mC mA -PEG- dT dG dA dT mC mC mU mG -3T 407       dC dA dGdG dC mU dA mC mG -PEG- dC dG dT dA mG dA mG dC +++ 16.36       dA mU dCmA -PEG- dT dG dA dT mC mC mU mG -3T 408       dC dA dG dG dC mU dA mCmG -PEG- dC dG dT dA mG dA mG dC +++ 28.4       dA mU mC mA -PEG- dT dGdA dT dC mC mU mG -3T 409       dC dA dG dG dC mU dA mC mG -PEG- dC dGdT dA mG dA mG dC +++ 7.313333       dA mU mC mA -PEG- dT dG dA dT mC dCmU mG -3T 410       dC dA dG dG dC mU dA mC mG -PEG- dC dG dT dA mG dAmG dC +++ 6.79       dA mU mC mA -PEG- dT dG dA dT mC mC dT mG -3T 513dC dC dC dA dG dG dC dT dA dC mG -PEG- dC dG dT dA mG dA mG dC ++++ 0.142.065 dA mU mC mA -PEG- dT dG dA dT mC dC dT mG mG mG -3T 514 dC dC dCdA dG dG dC dT dA dC mG -PEG- dC dG dT dA mG dA mG dC ++++ 0.13 3.155 dAmU mC mA -PEG- dT dG dA dT dC dC aT mG mG GG -3T 515 dC dC dC dA dG dGdC dT dA dC mG -PEG- dC dG dT dA mG dA mG dC ++++ 0.22 4.655 dA mU mC mA-PEG- dT dG dA dT dC dC dT dG dG dG -3T 516 dC dC dC dA dG dG dC dT dAdC mG -PEG- dC dG dT dA mG dA mG dC ++++ 0.14 3.045 dA mU mC mA -PEG- dTdG dA dT mC dC dT dG dG dG -3T

[0275] Following synthesis, these composition variants were tested in invitro biochemical binding and cell-based proliferation assays describedherein. These experiments showed a wide range of affinities in in vitrocompetition binding assays and a similarly wide range of activities incell-based proliferation assays (results are described below). Thecomposition variants that showed the highest levels of binding affinityand cell-based assay activity are exemplified by the series ARC513-516,shown in FIG. 6B. FIG. 6B shows the optimal composition variants ofARC126: ARC513 (5′-(SEQ ID NO:86)-PEG-(SEQ ID NO:57)-PEG-(SEQ IDNO:87)-3′ dT-3′), ARC514 (5′-(SEQ ID NO:86)-PEG-(SEQ ID NO:57)-PEG-(SEQID NO:88)-3′ dT-3′), ARC515 (5′-(SEQ ID NO:86)-PEG-(SEQ IDNO:57)-PEG-(SEQ ID NO:89)-3′ dT-3′), and ARC516 (5′-(SEQ IDNO:86)-PEG-(SEQ ID NO:57)-PEG-(SEQ ID NO:90)-3′ dT-3′). From FIG. 6B,all of the optimal composition variants are comprised of an extendedstem, relative to ARC126, by two base pairs, and in the upper stems, acommon set of point substitutions of 2′-O-methyl for 2′-fluoro residuesas well as the pre-existing 2′-O-methyl residues from ARC126. Thecomposition variants ARC513-516 differ only in two aspects: (1) thedeoxy or 2′-fluoro identity of the three 3′ terminal guanosine residuesin the central stem; and (2) the deoxy or 2′-O-methyl identity of thecytosine residue at the top of the central stem on the 3′ side.

[0276] The in vitro binding affinity of the optimal composition variantsfor PDGF is shown in FIG. 7A. The data shown in the figure was derivedfrom a competitive binding assay in which the indicated concentration ofunlabeled, competitor aptamer was titrated in separate reactions againsta fixed (<0.1 nM) amount of ³²P-radiolabeled ARC126 aptamer in buffercontaining a fixed amount (0.1 nM) of the aptamer's cognate target(PDGF-BB; PeproTech, Rocky Hill, N.J.). ARC126 was radiolabeled at the5′ terminus by incubation with γ-³²P-ATP (MP Biomedicals, Irvine,Calif.) and polynucleotide kinase (New England Biolabs (“NEB”), Beverly,Mass.). Binding reactions were carried out in phosphate-buffered saline(Cellgro, Herndon, Va.) containing 0.2 mg/mL bovine serum albumin (NEB)and 0.02 mg/mL tRNA (Sigma, St. Louis, Mo.). The reactions wereequilibrated for a period of 15-30 minutes at room temperature, thenfiltered through a sandwich of nitrocellulose (Protran membrane,Perkin-Elmer, Boston, Mass.) and nylon (Hybond membrane, Amersham,Piscataway, N.J.) membranes to separate target bound aptamer from freeaptamer. Subsequent auto-radiographic analysis of the filter membranescorresponding to each concentration of unlabeled aptamer revealed theextent of competitive displacement of ³²P-ARC126 by unlabeled aptamer.The data is shown in FIG. 7 wherein ARC128 ((5′-(SEQ ID NO:4)-HEG-(SEQID NO:5)-HEG-(SEQ ID NO:6)-3′)) represents a sequence-scrambled andtherefore inactive variant of ARC126.

[0277]FIG. 7B shows in vitro 3T3 cell-based proliferation assay datashowing the activity of some composition variants of ARC126. 3T3 cells,a rat fibroblast cell line (ATCC, Manassas, Va.), were plated 3,000cells/well in a 96 well plate one day prior to the start of the assay in100 ul DMEM/10%FCS. The following day, cells were washed once withstraight DMEM and then 75 ul DMEM/0.8% FCS was added to each well. Then25 ul of PDGF-BB (PeproTech, Rocky Hill, N.J.) at a final concentrationof 50 ng/ml±ARC126 variants (6 points, final concentration 0-200 nM)were added to each well. Cells were incubated for 3 days. Followingincubation 10 ul MTT (Promega, Madison, Wis.) was added to each well andincubated for an additional 1.5 hours. Media was then removed, 200 ulIsopropanol (2-propanol) was added to each well, cells were re-suspendedthoroughly and absorbance at 570 nm was read on a 96 well plate reader.As shown in FIG. 7B, it is clear that (1) all composition variants shownare active; and (2) the relative activity ranking of ARC513 (5′-(SEQ IDNO:86)-PEG-(SEQ ID NO:57)-PEG-(SEQ ID NO:87)-3′ dT-3′), ARC514 (5′-(SEQID NO:86)-PEG-(SEQ ID NO:57)-PEG-(SEQ ID NO:88)-3′ dT-3′), ARC515(5′-(SEQ ID NO:86)-PEG-(SEQ ID NO:57)-PEG-(SEQ ID NO:89)-3′ dT-3′), andARC516 (5′-(SEQ ID NO:86)-PEG-(SEQ ID NO:57)-PEG-(SEQ ID NO:90)-3′dT-3′) is similar.

[0278] Optimization of In Vivo Pharmacokinetic and BiodistributionProperties. In addition to optimization of the sequence composition ofARC126 with respect to target binding affinity and in vitro cell-basedassay activity, it is desirable to optimize the in vivo pharmacokinetic(PK) half-life, t_(1/2), and biodistribution properties of the optimalsequence composition(s) for the anti-PDGF aptamers described previously.This modulation of the pharmacokinetic and biodistribution properties ofan aptamer composition can be accomplished (see U.S. Ser No. 10/718,833,filed Nov. 21, 2003, and U.S. Ser. No. 60/550,790, filed Mar. 5, 2004)via conjugation of the 3′ or 5′ terminus, or an internal site or sites,of the molecule with a polyethylene glycol (PEG) chain or chains, i.e.,PEGylation (range is ˜2 kD to 100 kD, with typical PEGs having molecularweights of 2 kD, 10 kD, 20 kD, 30 kD, 40 kD, 50 kD).

[0279] In order to establish the feasibility of using a given PEGylatedaptamer sequence composition, it was necessary to confirm that theputative PEGylation does not significantly interfere with the activityof the aptamer in in vitro binding and cell-based proliferation assays.FIG. 8A shows competition binding assay curves for NH₂-ARC126(5′-amine-modified ARC126, no PEG) and two variants that are 5′conjugated to 30 kD (ARC308) and 40 kD (ARC127) PEG groups,respectively. Competitive binding assays were performed and analyzed asdescribed herein except that 3′-³²P-labeled NH₂-ARC126 was used ratherthan 5′-³²P-ARC126 (the ARC126 nucleotide sequence incorporates areverse thymidine at the 3′-terminus, which is a substrate for theradiolabeling reaction catalyzed by polynucleotide kinase).

[0280]FIG. 8A is a plot of competition binding assay data for ARC126 andtwo variants that are 5′ conjugated to 30 kD (ARC308) and 40 kD (ARC127)PEG groups. FIG. 8B shows in vitro 3T3 cell-based proliferation assaydata for ARC126 as a function of 5′ PEG group conjugation (ARC126+30 kD=ARC308, and ARC126+40 kD PEG=ARC127). The data shown in FIG. 8Bdemonstrate the 5′ conjugation of ARC126 to 30 kD and 40 kD PEG groupsappears to lead to a slight decrease in the in vitro activity of theaptamer, the effect of the presence of the PEG groups is less thantwo-fold relative to the unPEGylated aptamer, ARC126. The data shown inFIG. 8B also demonstrate that the 5′ conjugation of ARC126 to 30 kD and40 kD PEG groups does not significantly reduce the in vitro activity ofthe aptamer relative to the composition of ARC126.

[0281]FIG. 9A shows the in vivo pharmacokinetic profile of ARC126 as afunction of 5′ PEG group conjugation. Studies were done in CD-1 mice asdescribed in Example 10 (Charles River Labs, Wilmington, Mass.). Fromthe figure, it is clear that the terminal clearance half-life, t_(1/2),is strongly affected by the size of the 5′ PEG group conjugated to theaptamer.

[0282] The pharmacokinetic (PK) data shown in FIG. 9A was subjected tonon-compartmental analysis (NCA) using the industry-standard softwarepackage WinNonLin™ v.4.0 (Pharsight Corp., Mountain View, Calif.). Table3 below lists the primary NCA-derived in vivo pharmacokinetic (PK)parameters for ARC126+20 kD (ARC240), +30 kD (ARC308), and +40 kD(ARC127) PEG groups after intravenous (IV) administration at 10 mg/kg inmice. The data shown in Table 3 demonstrate that the in vivopharmacokinetic clearance half-life, t_(1/2), of ARC126 is modulated≧4-fold by changing the size of the 5′ PEG group conjugated to theaptamer affected by the size of the 5′ PEG group conjugated to theaptamer from 20 kD to 40 kD. FIG. 9B shows the in vivo pharmacokineticprofile of ARC127 (ARC126+40 kD PEG) after intravenous (IV),intraperitoneal (IP), and subcutaneous (SC) administration at a doselevel of 10 mg/kg in mice. The pharmacokinetic (PK) data shown in FIG.9B was subjected to noncompartmental analysis (NCA) using theindustry-standard software package WinNonLin™ v.4.0 (Pharsight Corp.,Mountain View, Calif.). TABLE 3 Pharmacokinetic non-compartmentalanalysis of 5′ conjugates of ARC126. AUC, PEG Cmax, nM nM · hr t_(max)(hr) MRT (hr) t_(1/2) (hr) V_(ss) (mL/kg) ARC240 20K 34317 0.5 392771.16 0.94 0.3 ARC308 30K 27659 1.0 64867 2.14 1.67 0.3 ARC127 40K 609642.0 344356 5.08 6.84 0.1

[0283] Table 4 below lists the primary NCA-derived in vivopharmacokinetic (PK) parameters for ARC126+40 kD PEG as a function ofthe route of administration at 10 mg/kg in mice. The pharmacokinetic(PK) data shown in FIG. 9B was subjected to noncompartmental analysis(NCA) using the industry-standard software package WinNonLin™ v.4.0(Pharsight Corp., Mountain View, Calif.). TABLE 4 Pharmacokineticprofile of ARC127 (ARC126 + 40 kD PEG) Cmax, nM t_(max) (hr) AUC (hr ·nM) MRT (hr) t_(1/2) (hr) V_(z) (L/kg) bioavailability, F IV 29711.6 2229686.8  6.573 8.602 0.053 1.000 IP 12756.0 8 143605.5 11.231 7.8560.078 0.625 SC 3176.7 8 55030.91 16.632 9.176 0.238 0.240

[0284] From data shown in FIG. 9B, two primary points are clear: (1) thepharmacokinetics of ARC127 (ARC126+40 kD PEG) after intravenous (IV),intraperitoneal (IP), or subcutaneous (SC) administration at a doselevel of 10 mg/kg in mice a plasma concentration of ˜1 μM is present at24 hrs post-dose; and (2) the systemic bioavailability, F, of ARC127after intraperitoneal (IP) administration is quite high (˜63%), whilefor subcutaneous (SC) administration the bioavailability is still highenough (˜24%) to warrant consideration for clinical applications.

[0285] As a secondary test of both the plasma pharmacokinetics and thein vivo bioactivity of ARC127 (ARC126+40 kD PEG), the competitionbinding assay described above in reference to FIG. 7 was used to assaythe same plasma samples used to generate the data shown in FIG. 9B.Serial 1:10 dilutions of plasma sample were prepared inphosphate-buffered saline (1×PBS) and mixed with a fixed concentrationof ³²P-ARC126, then added to human PDGF-BB. The final concentration ofPDGF in each assay was 0.1 nM, and the final concentration of³²P-ARC126<0.1 nM. In this experiment, the plasma samples were analyzedby comparison with a standard curve generated with samples of knownARC127 concentrations in 1×PBS. By comparison with the referencestandards, the effective concentration of active aptamer in each plasmasample could be calculated. The effective concentration of activeaptamer, as calculated using the results of the competition bindingassay analysis of the plasma PK samples, is shown in FIG. 9C. FIG. 9Cshows the bioactivity profile of ARC126+40 kD PEG after intravenous (IV)administration at a dose level of 10 mg/kg in mice. This ex vivoanalysis thus provides verification that (1) the aptamer was present andactive in the plasma of the mouse model at t=48 hrs post-dose; and (2)the plasma concentrations calculated from the fluorescence-basedpharmacokinetic assay are correct.

Example 4 Species Cross Reactivity of ARC126 and ARC127

[0286] Studies were performed to determine which isoforms of PDGF ARC126would bind to. Competition binding assays were set up using the dot blotanalysis previously described, to test ARC126 for binding to humanPDGF-AA, PDGF-BB and PDGF-AB (all from PeproTech, Rocky Hill, N.J.). Theresults of the competition binding assay show that ARC126 (SEQ ID No. 4)binds to PDGF-BB with a K_(d) of approximately 100 pM, and PDGF-AB witha K_(d) of approximately 100 pM, but does not bind to PDGF-AA (FIG.10A). Next, a study was done to determine whether ARC126 cross reactedwith PDGF-BB of species other than human. Competition binding assayswere set up by dot blot analysis, as previously described, using human,rat, and mouse PDGF-BB (PeproTech, Rocky Hill, N.J.). The results of thecompetition binding assay show that ARC126 binds to human, rat and mousePDGF-BB with equal affinity (FIG. 10B).

Example 5 3T3 Cell Proliferation Assay

[0287] Since ARC126 was shown to cross react with human, rat and mousePDGF-BB, 3T3 cells, which are derived from a rat fibroblast cell line(ATCC, Manassas, Va.), could be used in a proliferation assay to testpotency of all PDGF aptamers, including aptamers that were obtained aspart of the de-fluorination efforts. 3T3 fibroblast cells have PDGFreceptors on their cell-surface and respond to mitogen, e.g., PDGFstimulation by proliferation. The assay was performed as follows: 3T3cells were plated 3,000 cells/well in a 96 well plate one day prior tothe start of the assay in 100 ul DMEM/10% FCS. The following day, cellswere washed once with straight DMEM and then 75 ul DMEM/0.8% FCS wasadded to each well. Then 25 ul of PDGF-BB (PeproTech, Rocky Hill, N.J.)at a 50 ng/ml final concentration was added to each well±aptamercondition to be tested. Each plate included the following conditions intriplicate: no PDGF which corresponds to growth without mitogen(negative control), a scrambled aptamer control (ARC128) where no effecton growth rate is observed (negative control), a positive control wheremaximal growth is observed in the absence of PDGF aptamer, and a seriesof functional PDGF aptamer dilutions from which a good IC50 curve couldbe calculated. The functional PDGF aptamer dilutions usually consists of6 points in 2-fold serial dilutions.

[0288] Cells were incubated for 3 days. Following incubation 10 ul MTT(Promega, Madison, Wis.) was added to each well and incubated for anadditional 1.5 hours. Media was removed, 200 μl Isopropanol (2-propanol)was added to each well, cells were re-suspended thoroughly andabsorbance at 570 nm was read on a 96 well plate reader. The presentexample depicts the potency comparison of PDGF aptamer ARC 127 to thatof a PDGF neutralizing polyclonal antibody as shown in FIG. 11A (R&DSystems, Minneapolis, Minn.). The data shown in FIG. 11A demonstratethat the aptamer displays better potency than the polyclonal antibody.

[0289] The 3T3 cell proliferation assay was performed with ARC126,ARC127, ARC128 and with all the other PDGF aptamer derivatives that wereobtained as described in Example 1 above. ARC126 and ARC127 routinelydisplay IC₅₀ values <20 nM where scrambled control ARC128 never displaysan effect of 3T3 cell proliferation. Table 5 below shows the IC₅₀s ofARC126 variants in 3T3 proliferation assay. TABLE 5 IC50's ofdefluorinated ARC126 variants proliferation assay. Mean ARC# IC50 12420.67 126 3.40 127 3.45 276 18.00 277 8.67 299 1000.00 300 900.00 33590.00 336 196.67 337 1000.00 338 1000.00 339 766.67 340 866.67 3411000.00 343 342 1000.00 344 200.00 345 13.00 346 15.00 347 1000.00 362315.00 363 3.92 364 1719.00 365 6.79 366 5.54 404 3.89 405 8.65 40617.15 407 16.36 408 28.40 409 7.31 410 6.79 513 2.07 514 3.16 515 4.66516 3.05 127 3.45

[0290] The ARC 127 PDGF aptamer also blocks proliferation of 3T3 cellsbetter than known tyrosine kinase inhibitors such as AG 1295 and AG 1433compounds (Sigma Aldrich Biochemicals, St Louis, Mo.). The assayconditions were exactly the same as described above. ARC127 reduced thePDGF driven increase in proliferation to background levels at aconcentration as low as 30 nM. Both of the AG compounds displayed muchworse potencies compared to ARC127. AG-1433 seemed to have unspecifictoxic effects at micromolar levels. This effect is visible starting from300 nM where signal levels are lower than no treatment alone samplescorresponding to loss of signal due to lethality of cells only in thepresence of AG compound (FIG. 11B).

Example 6 3T3 Cell Viability Assay

[0291] The reduction of growth in 3T3 cells observed in the cellproliferation assay described in Example 5 upon addition of ARC126,ARC127 and other active PDGF aptamer derivatives might potentially bedue to toxic effects of aptamer. To test this possibility a Calcein AMcell viability assay (Molecular Probes, Eugene, Oreg.) was performed.3T3 cells were plated 3,000 cells/well were treated with variousconcentrations of PDGF aptamer up to 40 μM were tested for 24 and 48hours. TNF alpha (100 pg/ml) was provided and used as a positive controlto induce apoptosis. Following incubation cells were washed with 1×PBS.Calcein AM was prepared according to manufacturer's recommendedinstructions, incubated for 30 minutes and fluorescence signal intensitywas determined on a 96-well plate reader. No increase in the apoptosisrate of 3T3 cells due to ARC127 was observed (FIG. 12).

Example 7 3T3 or RPE Cell Migration Assay

[0292] PDGF is a strong mitogen as well as a chemoattractant. Amigration assay performed both in 24 and 96-well format was chosen as anadditional functional assay to test the potency of ARC127. In the cellmigration assay 80,000 cells 3T3 rat fibroblasts (ATCC, Manassas, Va.)or RPE (retinal pigmented epithelial) cells (ATCC, Manassas, Va.) wereplated per well into a 24 well plate with 8 micron filters (BDBiosciences, Discovery Labware, Bedford, Mass.). 0.5 ml DMEM/0.2% FCSwas added to the top chamber and 0.8 ml DMEM/0.2% FCS was added to thebottom chamber. The system was equilibrated by incubating for 4 hours at37 degrees. Human PDGF-BB (PeproTech, Rocky Hill, N.J.) (for RPE cells)or rat PDGF-BB (PeproTech) (for 3T3 cells) was added to the bottomchamber (0 ng/ml-100 ng/ml final concentration). The system wasincubated 4 hours to 12 hours. Cells were scraped off on the top offilter with a Q-tip. The cells that migrated to the bottom of the filterwere fixed with a mixture of cold 50% Methanol/50% Acetone for 3minutes.

[0293] Following incubation filters were washed with 1×PBS and stainedwith Giemsa Stain (Matheson Coleman and Bell) for 1-2 hours andmigration was visualized by taking pictures on a Zeiss Axiovert 200Mmicroscope. Specifically, migration under four different conditions wasvisualized: (1) background migration observed in the absence of PDGF;(2) migration observed in the presence of 5 nM PDGF BB; (3) migrationobserved in the presence of 5 nM PDGF BB and 100 nM functional aptamerARC127; and (4) migration observed in the presence of 5 nM of PDGF BBand 100 nM ARC128 scrambled control aptamer. The visualized migrationresults show that the presence of 100 nM ARC127 inhibits the effects of5 nM PDGF-BB, shown by the migration of 3T3 or RPE cells at backgroundlevels. ARC128 scrambled control displays no activity and the migrationobserved at this condition is equal to the one observed with 5 nM PDGFBB alone.

[0294]FIG. 13 shows the results of a cell migration experiment performedin 96 well format using QCM Chemotaxis 96 Well Migration Assay (#ECM510) (Chemicon, Temecula, Calif.). The 96 well format allowed for a morequantitative analysis of cell migration than the 24 well format, whichwas more qualitative. The 96 well assay starts by bringing plates andreagents to room temperature. 150 ul of DMEM/0.2% FBS was added with orwithout chemoattractant to the wells of the feeder tray. 200,000 cellsRPE cells in 100 ul media DMEM/0.2 FBS were added into migration chamberand incubated for 1-24 hours at 37 degrees.

[0295] Following incubation, the migration chamber plate was removed andthe non-migratory cells were discarded. The number of migratory cellswas quantitated according to manufacturer's recommended instructions. Inbrief, media from the top side was removed by flipping out the remainingcell suspension, and was placed onto a new feeder tray that contains 150ul of pre-warmed Cell Detachment Solution. This mixture was incubatedfor 30 minutes at 37 degrees. CyQuant GR Dye 1:75 was diluted with 4×Lysis Buffer and 50 ul of the Lysis Buffer/Dye Solution was added toeach well containing 150 ul of the solution containing migratory cells.This mixture was further incubated for 15 minutes at room temperaturethen transferred to a fresh 96 well plate and read at 480/520 nmabsorbance on a 96 well plate reader.

[0296] The results obtained in this 96 well format cell migrationexperiment (FIG. 13) confirmed the cell migration experiments done in 24well format. ARC127 reduced migration levels to background and ARC128scrambled aptamer did not affect migration. FIG. 13 also shows that themigration observed increases linearly as a function of cell number orconcentration of mitogen added.

Example 8 Clonogenic Growth Assay

[0297] U87 glioblastoma cells (ATCC) were grown in the presence orabsence of mitogen as well as aptamer as shown in the panels. U87 cellswere plated in DMEM/10% FBS to 50% confluency in 100 mm dishes. Cellswere incubated with PDGF-BB (PeproTech)±ARC127 until cells appearedconfluent (1-2 days). The addition of a final concentration of 50 ng/mlPDGF BB alone caused the appearance of highly connected threedimensional cell clusters. Addition of 50 ng/ml PDGF-BB plus 10 nM to100 nM functional aptamer ARC127 reduced the occurrence of clusters tobackground level. The presence of aptamer had no effect on proliferationrate of the cells determined by MTT assay. Thus, the aptamer blocks cellto cell adhesion of U87 cells which are known to have PDGF drivenautocrine loops. ARC127 seems to be blocking the cell surface displayedligand binding to another cell's receptor as displayed by cell to celladhesion.

Example 9 PDGF-Drive ELK Luciferase Assay

[0298] To further prove the activity of ARC127 PDGF aptamer, amechanistic Elk Luciferase reporter gene assays was setup. 10,000 3T3cells/well were plated in DMEM/10% FBS in a 96 well plate. They weretransfected using FuGene (Roche, Indianapolis, Ind.) at 3:1 ratio with 5ng of ELK-1 (Stratagene, La Jolla, Calif.) and 20 ng of pFR-Luciferaseplasmids (Stratagene, La Jolla, Calif.). When PDGF is added at a finalconcentration of 50 ng/ml to 3T3 cells, an increase in the Luciferasesignal can be observed which corresponds to the effect of the mitogen onthe reporter gene. Steady Glo Luciferase assay (Promega, Madison, Wis.)was used to detect luciferase. The results indicate that a concentrationof ARC127 as low as 3 nM reduces the Luciferase signal to no mitogenlevels. IC50 of ARC127 deduced from this analysis is <2 nM. FIG. 14.

Example 10 In Vivo Data in HT-29 and LS174T Colon Cancer XenograftModels

[0299] In vivo efficacy studies were established to test the hypothesisthat inhibition of PDGF-BB and/or its receptor with ARC127 and ARC308(increase the efficacy of cytotoxic drugs.

[0300] Experimental overview. Human HT-29 or LS174T colon cancerxenotransplants were established in athymic nude mice (Nu/Nu mice,Charles River Labs, Wilmington, Mass.) by injecting mice with 1×10⁷ HT29cells (ATCC, Manassas, Va.), or 2×10⁶ LS174T cells (ATCC Manassas, Va.)sub-cutaneously and allowing the tumors to grow for 5 days. On day 5,two dimensional measurements of the established tumors were taken usinga digital caliper. Once tumor measurements were taken, the mice wererandomized into groups such that the average tumor size was the same ineach group. Once randomized, the mice were treated with irinotecan(Pfizer, NY, N.Y.) which is a cytotoxic drug shown to have efficacyagainst colon cancer in the presence or absence of PDGF blockade using aPDGF specific aptamer or other small molecule inhibitor, to determine ifPDGF blockade increases the efficacy of the cytotoxic drug.

[0301] Materials and Methods. An experiment (Experiment 1) was designedto test a combination of known chemotherapeutic agents GLEEVEC™ andirinotecan in a dose optimization study in an HT29 colon cancerxenograft model. Table 6 below summarizes the experimental design ofExperiment 1 using a human colon carcinoma cell line HT29 (ATCC,Manassas, Va.), with a cytotoxic drug, irinotecan (Pfizer, NY, N.Y.)administered at 150 mg/kg via intra-peritoneal injection once weekly, aswell as a drug to block PDGF signaling, GLEEVEC™ (Novartis, Basel,Switzerland) dosed orally at 50 mg/kg twice daily (Monday throughFriday). Though the mode of action of GLEEVEC™ is known to block thePDGF receptor function as well as other receptors for other growthfactors, the effect is not necessarily PDGF specific. The results ofthis experiment are shown in FIG. 1SA showing a plot of group meanaverage tumor diameter in mm as a function of time for each of thetreatment regimens irinotecan 150 mg/kg weekly, GLEEVEC™ 50 mg/kg orallyBID Qd5, and irinotecan 150 mg/GLEEVEC™ 50 mg/kg BID Qd5 combinationtherapies. These data show that GLEEVEC™ increases the efficacy ofirinotecan alone in HT29 colon cancer xenotransplant model.GLEEVEC™-mediated PDGF blockade enhanced the efficacy of irinotecantreatment as demonstrated by the decreased rate of tumor growth ascompared to animals treated with irinotecan alone. The results arestatistically significant (two tailed students T-test). TABLE 6Experiment 1 HT29 irinotecan/GLEEVEC Dose Optimization Study AnimalStudy Number: 03003-005 Proposed Start Date: Jan. 13, 2003 CombinationTherapy Administration (PLEASE DOSE AFTER TEST Tumor Inoculation TestArticle Administration ARTICLE) No. of Test Test Dose Test Dose Day ofGroup Animals Material Dose Route Day Material (mg/kg) Route DayMaterial (mg/kg) Route Day Euthanasia 1 10 HT-29 1 × 10⁷ SC Day 0Diluent DVE IP Days NA NA IP SID TBD 2 10 cells Diluent DVE 7, 14, ARC127  50 Days 3 10 Irinotecan 150 21 NA NA 4-21 4 10 Irinotecan  50 NA NA5 10 Irinotecan 150 ARC 127  50 6 10 Irinotecan  50 ARC 127  50 7 10Irinotecan 150 Gleevec 100 PO 8 10 Irinotecan  50 Gleevec 100 9 DiluentDVE Gleevec 100

[0302] Another experiment (Experiment 2) was designed to test ARC127 andirinotecan in a colon cancer xenograft model using human colon carcinomacell line LS174T (ATCC, Manassas, Va.). Cytotoxic drug, irinotecan(Pfizer, NY, N.Y.) was dosed once weekly at 150 mg/kg) viaintraperitoneal delivery. ARC127 used to block PDGF signaling, was dosedat 50 mg/kg via intra-peritoneal delivery once daily, and GLEEVEC™(Novartis, Basel, Switzerland) was dosed orally at 100 mg/kg once daily,Monday through Friday. ARC127 prevents PDGF-BB from binding the PDGFreceptor; and has a 40K PEG attached. Table 7 below summarizes theexperimental design for Experiment 2. The results from Experiment 2 areshown in FIG. 15B demonstrating that ARC127 enhanced the efficacy ofirinotecan treatment as demonstrated by the decreased rate of tumorgrowth as compared to animals treated with irinotecan alone. The resultsare statistically significant (two tailed students T-test). The dataclearly show that ARC127 increases the efficacy of irinotecan betterthan both GLEEVEC™/irinotecan combination treatment and irinotecan alonein LS174T colon cancer xenotransplant model. The GLEEVEC™ dosing regimen(100 mg/kg once daily, Monday through Friday, P.O.) made the animalsmoribund and these groups were terminated early in the experiment (FIG.15B). TABLE 7 ARC127/irinotecan dosing study in LS174T colon cancerxenograft model Experimental Design: Animal Study Number: 03003-007Proposed Start Date: Feb. 5, 2003 Combination Therapy Administration(PLEASE DOSE AFTER TEST Tumor Inoculation Test Article AdministrationARTICLE) No. of Test Test Dose Test Dose Day of Group Animals MaterialDose Route Day Material (mg/kg) Route Day Material (mg/kg) Route DayEuthanasia 1 10 LS174T 2 × 10⁶ SC Day Diluent DVE IP Days NA NA IP SIDTBD 2 10 cells 0 Diluent DVE 8, 15, ARC 127  50 Days 3 10 Irinotecan 15022 NA NA 4-21 4 10 Irinotecan 150 ARC 127  50 5 10 Irinotecan 150Gleevec 100 PO 6 10 Diluent DVE Gleevec 100

[0303] A third experiment (Experiment 3) was designed to testARC308/irinotecan and GLEEVEC™/irinotecan dosing regimens in a coloncancer xenotransplant model using human colon carcinoma cell line LS174T(ATCC). Cytotoxic drug irinotecan (Pfizer, NY, N.Y.) was dosed viaintraperitoneal delivery at 150 mg/kg once weekly. ARC308 was dosed viaintraperitoneal delivery at 50 mg/kg once daily, and GLEEVEC™ was dosedat 50 mg/kg once daily, Monday through Friday. ARC308 prevents PDGF-BBfrom binding the PDGF receptor; this molecule has a 30K PEG attached.The results from Experiment 3 are shown in FIG. 16 showing that ARC 308enhanced the efficacy of irinotecan treatment as demonstrated by thedecreased rate of tumor growth as compared to animals treated withirinotecan alone (FIG. 16). In contrast the GLEEVEC™ dosing regimen (50mg/kg once daily, Monday through Friday, P.O) did not enhance theefficacy of Irinotecan. The results are statistically significant (twotailed students T-test). Table 8 summarizes the experimental design forExperiment 3. TABLE 8 ARC308/irinotecan and GLEEVEC/irinotecan studyLS174T colon cancer xenograft model. Animal Study Number: 03003-014Proposed Start Date: Mar. 4, 2004 Combination Therapy Administration(PLEASE DOSE AFTER TEST Tumor Inoculation Test Article AdministrationARTICLE) No. of Test Test Dose Test Dose Day of Group Animals MaterialDose Route Day Material (mg/kg) Route Day Material (mg/kg) Route DayEuthanasia 1 10 LS174T 2 × 10⁶ SC Day Diluent DVE IP Days NA NA IP SIDTBD 2 10 cells 0 Diluent DVE 8, 15, ARC 308 50 Days 3 10 Irinotecan 15022 NA NA 5-22 4 10 Irinotecan 150 ARC 308 50 5 10 Irinotecan 150 Gleevec50 PO M-F 6 10 Diluent DVE Gleevec 50

[0304] In summary, these in vivo studies in HT-29 and LS174T coloncancer xenograft models confirm that PDGF blockade effected by thetherapeutic aptamers of the present invention can increase the efficacyof irinotecan treatment.

Example 11 PDGF/VEGF Bi-Functional Aptamers as Oncology Therapeutics

[0305] Combination therapies have advantages over single-agent therapiesin the treatment of solid tumors as shown in the colon cancer xenograftmodels with the aptamers of the present invention (Example 10).Similarly, aptamers that are able to bind to more than one of thetargets that are implicated in solid tumor cancers are also effective inpotentiating the therapeutic effects of individual therapeutic agents.Aptamers such as these can inhibit multiple proteins (or other targets)and therefore provide a single compound that acts in a manner that issubstantially equivalent to a combination of compounds. Multi-functionalaptamers can be engineered, e.g., from combinations of known aptamers.These multifunctional aptamers can be shown to bind to multiple targets,and can be generated either directly by solid-phase chemical synthesis,or by transcription from a corresponding DNA template. Examples of suchmulti-functional aptamers include aptamers capable of binding to VEGFand PDGF for cancer indications.

[0306] In order to design multifunctional aptamers from previouslyidentified aptamers (or regions of aptamers) it is important to conjointhe individual aptamers via regions of the individual aptamer that donot make contact with the target. This can typically be accomplished byidentifying regions of the secondary structure which toleratesubstitution of individual nucleotides at most or all positions. Ifstructural units are required, such as a stem, then these can bepreserved in the final design. Additionally, it is important that thestructural integrity of each of the individual aptamers is preserved inthe final folded structure. This can most easily be achieved bypredicting the secondary structures of the original aptamer sequencesusing an algorithm such as mfold, and then ensuring that these predictedsecondary structures are preserved, according to the same algorithm,when they are part of the conjoined structure. The general mfoldalgorithm for determining multiple optimal and suboptimal secondarystructures is described by the author of the program, Dr. Michael Zuker(Science 244, 48-52 (1989)). A description of the folding parametersused in the algorithm is presented in Jaeger, Turner, and Zuker, Proc.Natl. Acad. Sci. USA, (1989) 86, 7706-7710 (see also M. Zuker, et al.,Algorithms and Thermodynamics for RNA Secondary Structure Prediction: APractical Guide in RNA Biochemistry and Biotechnology, J. Barciszewskiand B. F. C. Clark, eds., NATO ASI Series, Kluwer Academic Publishers,(1999)). Other programs that can be used according to the methods of thepresent invention to obtain secondary structures of nucleic acidsequences include, without limitation, RNAStructure (Mathews, D. H.;Sabina, J.; Zuker, M.; and Turner, D. H., “Expanded Sequence Dependenceof Thermodynamic Parameters Improves Prediction of RNA SecondaryStructure,” Journal of Molecular Biology, 288, 911-940(1999), and RnaViz(Nucleic Acids Res., November 15; 25(22):4679-84 (1997).

[0307] Having determined the secondary structural motifs and units ofthe component aptamers, these are combined into a single oligonucleotidethat folds in a manner that preserves the functionalities of each of theconstituent aptamers. As a result, multifunctional aptamers are capableof binding to multiple targets, and can be generated either directly bysolid phase chemical synthesis, or by transcription from a correspondingDNA template.

[0308] VEGF and PDGF Bifunctional Aptamers. The methods of the presentinvention were applied to generate an aptamer having binding specificityto PDGF BB and to VEGF. This multi-functional aptamer was generated byjoining two aptamers individually identified using SELEX-one (ARC 245,SEQ ID No. 7) which recognized VEGF but not PDGF (see FIG. 17A) and one(ARC 126, 5′-(SEQ ID NO:1)-HEG-(SEQ ID NO:2)-HEG-(SEQ ID NO:3)-3′ dT-3′)which recognized PDGF but not VEGF (see FIG. 17B). ARC245 (SEQ ID No. 7)is a fully 2′O methylated (2′-OMe) aptamer with binding specificity toVEGF with a K_(D) of about 2 nM. The ARC126 used in the multivalentaptamer is entirely DNA with binding specificity to PDGF with a K_(D) ofabout 100 pM.

[0309] A schematic of the structure and sequence of the multivalentaptamer capable of binding to PDGF and VEGF resulting from thiscombination, sequence TK.131.012.A (SEQ ID No. 9), is shown in FIG. 18A.A second multivalent aptamer capable of binding to VEGF and PDGF,sequence TK.131.012.B (SEQ ID No. 10), was also formed by combiningARC245 (SEQ ID No. 7) and ARC 126. As shown in FIG. 18B, in thisVEGF-PDGF multivalent aptamer the first mA-mU pair of the stem of ARC245was removed before joining ARC245 and all deoxy-ARC126. In each case, asshown in FIG. 18, one of the ARC126 PEG linkers was removed for theaddition of the VEGF specific aptamer and the other was substituted withan oligonucleotide linker having the sequence dGdAdAdA.

[0310] Binding data for the constituent aptamers and the multivalentaptamers were collected by dot-blot assays in which radio-labeledaptamer is incubated with protein target and then forced through asandwich of nitrocellulose over nylon and the results shown in FIG. 19.Protein-associated radio-label is captured on the nitrocellulosemembrane while the balance of the radio-label is captured on the nylonmembrane. The radio-label data is collected on a phosphorimaging plate.This data is then used to calculate the binding coefficients. Themultivalent aptamers TK.131.012.A (SEQ ID No. 9) and TK.131.012.B (SEQID No. 10) were made synthetically.

[0311] The multivalent aptamers with the sequence TK.131.012.A (SEQ IDNo.9) shows a K_(D) of about 10 nM for PDGF and about 10 nM for VEGF.The multivalent aptamer with the sequence TK.131.012.B (SEQ ID No. 10)shows a K_(D) of about 10 nM for PDGF and about 5 nM for VEGF.

Example 12 PDGF and VEGF Aptamers Containing Immunostimulatory Motifs

[0312] To test the ability of aptamers comprising CpG motif(s) tostimulate the innate immune response, a murine CpG motif was engineeredinto an aptamer specific for PDGF-B. These aptamers were then used in invitro mouse cell-based assays to confirm functionality of the CpG motifs(e.g., stimulation of the release of IL-6 and TNF-alpha). These aptamerswere also used in aptamer mechanism-based biological activity assays(PDGF-B signaling through the MAPK pathway as measured by the effect ofthe CpG-PDGF-B-aptamer on PDGF-B activation of ERK Phosphorylation) toconfirm functionality of the CpG motif comprising aptamer upon bindingof the aptamer to its target, in this case PDGF-B.

[0313] To generate the aptamers disclosed herein with an embedded orappended CpG motif, the sequence of previously identifiedimmunostimulatory oligonucleotides comprising CpG motifs (“ISS-ODN” or“ODN”) or fragments thereof were engineered into ARC124, an aptameridentified through the SELEX process that binds to PDGF AB and BB with aK_(d) of approximately 100 pm. The sequence of ARC124 is shown below.ARC124 (SEQ ID NO.: 11) 5′CACAGGCTACGGCACGTAGAGCATCACCATGATCCTGTG3′InvdT

[0314] The various ODN sequences, both full length and fragments orderivatives thereof are shown below 5′→3′ from left to right (whereinan * indicates a phosphorothioate bond and 3InvdT indicates an invertedT at the 3′ end). In addition to these ODN sequences or fragmentsthereof being engineered into PDGF aptamers, they were also used forcontrols in assaying the ability of these aptamers to stimulate theimmune response. ISS-ODN (SEQ ID NO.: 12) T*G*A* C*T*G* T*G*A* A*C*G*T*T*C* G*A*G* A*T*G* A ISS-ODN2 (SEQ ID NO.: 13) T*G*A* A*C*G* T*T*C*G*A*G* A*T* ISS-ODN3 (SEQ ID NO.: 14) A* A*C*G* T*T*C* G*A*G* A*T*ISS-ODN4 (SEQ ID NO. 15) A* A*C*G* T*T*C* G*A*G ISS-ODN5 (SEQ ID NO.:16) G*T*G* A*A*C* G*T*T* C*G*A* G ODN 2006 (SEQ ID NO.: 17) T*C*G*T*C*G* T*T*T* T*G*T* C*G*T* T*T*T* G*T*C* G*T*T ODN 2006.2 (SEQ ID NO.:18) G* T*C*G* T*T*T* T*G*T* C*G*T* T*T*T* G*T ODN 2006.3 (SEQ ID NO.:19) G* T*C*G* T*T*T* T*G*T* C*G*T* T

[0315] ISS-ODN was identified from Martin-Orzco et al., Int. Immunol.,1999. 11(7): 1111-1118. ISS-ODN 2-5 are fragments of ISS-ODN. ODN 2006was identified from Hartmann et al., Eur. J. Immunol. 2003.33:1633-1641. ODN 2006.2 and 2006.3 are fragments of ODN 2006.

[0316] The sequences of the PDGF aptamers comprising a CpG motif areshown below 5′→3′ from left to right (wherein an * indicates aphosphorothioate bond and 3InvdT indicates an inverted T at the 3′ end).A schematic of the sequence and secondary structure of these aptamers isshown in FIG. 20. CpGARC124short a.k.a. shortARC124 (SEQ ID NO.: 20)A*A*C* G*T*T* C*G*A* G* CA GGC TAC GGC ACG TAG AGC ATC ACC ATG ATC CT*G*C/3InvdT/ LongCpGARC124 a.k.a. longARC124 (SEQ ID NO.: 21) G*T*G* A*A*C*G*T*T* C*G*A* G* CA GGC TAC GGC ACG TAG AGC ATC ACC ATG ATC CT*G*C/3InvdT/ FullCpGARC124 also referred to herein as fullARC124 (SEQ IDNO.: 22) T*G*A* C*T*G* T*G*A* A*C*G* T*T*C* G*A*G* A*T*G* A* CA GGC TACGGC ACG TAG AGC ATC ACC ATG ATC CT*G* T*T*T* T*T*T* T TransARC124.1 (SEQID NO.: 23) C*A*G*GCTAC*G*T*T*C*GTAGAGCATCACCATGATC*C*T*G*/3InvdT/TransARC124.2 (SEQ ID NO.: 24)C*A*G*GCTAC*G*T*T*T*C*GTAGAGCATCACCATGATC*C*T*G*/3InvdT/ TransARC124.3(SEQ ID NO.: 25)C*A*G*GCAAC*G*T*T*T*C*GTTGAGCATCACCATGATC*C*T*G*/3InvdT/ TransARC124.4(SEQ ID NO.: 26) C*A*G*GCAAC*G*T*T*C*GTTGAGCATCACCATGATC*C*T*G*/3InvdT/TransARC124.5 (SEQ ID NO.: 27)C*A*G*GCAAC*G*T*T*T*T*C*GTTGAGCATCACCATGATC*C*T*G*/3InvdT/ TransARC124.6(SEQ ID NO.: 28) C*A*G*GCTACGTTTCGTAGAGCATCACCATGATC*C*T*G*/3InvdT/TransARC124.7 (SEQ ID NO.: 29)C*A*GGCTACGTTTCGTAGAGCATCACCATGATCC*T*G*/3InvdT/ TransARC124.8 (SEQ IDNO.: 30) C*A*G*GCGTCGTTTTCGACGAGCATCACCATGATC*C*T*G*/3InvdT/TransARC124.9 (SEQ ID NO.: 31)C*A*G*GCGTCGTCGTCGACGAGCATCACCATGATC*C*T*G*/3InvdT/ TransARC124.10 (SEQID NO.: 32) C*A*G*GCTTCGTCGTCGAAGAGCATCACCATGATC*C*T*G*/3InvdT/TransARC124.11 (SEQ ID NO.: 33)C*A*G*GCTACGTCGTCGTAGAGCATCACCATGATC*C*T*G*/3InvdT/

[0317] As indicated above, ISS-ODN and ODN 2006 are phosphorothioatedoligonucleotides that are reported to be immunostimulatory and theirsequences are derived from the literature. Truncated versions of theseoligonucleotides were designed by shortening them from both their 5′ and3′ ends. The resulting constructs are ISS-ODN2 through 5 and ODN 2006.2through 3. Each of the newly designed constructs was tested in IL-6release assay to assess the effect on immunostimulatory ability. Theshortest construct that still retained the ability to induce IL-6release was picked for the sequence appended either to the 5′ or 3′ endof ARC124. Constructs that are labeled as CpGARC124short a.k.a.shortARC124, LongCpGARC124 a.k.a. longARC124, and FullCpGARC124 a.k.a.fullARC124 correspond to constructs where ISS-ODN4 is appended to the5′-end of ARC124. These constructs carry phosphorothioated bonds attheir 5′ and 3′ ends in addition to inverted T at their 3′-end to assurethe adequate stability in the cell-based assay but the core middle partcorresponding to ARC124 is free of phosphorothioate bonds.

[0318] ARC124 at position 8-9 and 13-14 has two naturally occurring CpGislands. Constructs with the nomenclature of TransARC124.1 throughTransARC124.11 correspond to constructs that made use of these twonaturally occurring CpG sequences to create an immunostimulatorysequence that would maximize the CpG response based on reports in theliterature that CpG motifs that are embedded in varying lengths of Tscreate maximal immunostimulatory effect. Thus, the changes that wereperformed on ARC124 consisted of substituting the non-essential GCAbulge at position 10-12 with various T residues. The effect of varyingthe T bulge on the immunostimulatory effect as well as addition ofphosphorothioated bonds on the stability of the construct are assessedwith IL-6 release assay and phosphorylation of ERK as described herein.

[0319] For negative controls, ARC124 was engineered to remove the CpGmotifs. These control sequences are shown below. TransARC124.3control(CpG-Scrambled Aptamer) (SEQ ID NO.: 34)C*A*G*GCAAG*C*T*T*T*G*CTTGAGCATCACCATGATC*C*T*G*/3InvdT/TransARC124.5control (CpG-Scrambled Aptamer) (SEQ ID NO.: 35)C*A*G*GCAAG*C*T*T*T*T*G*CTTGAGCATCACCATGATC*C*T*G*/3InvdT

[0320] The activity of PDGF aptamers containing CpG islands of thepresent invention were tested in cell based assays. Supernatants ofJ774A.1 cells (TIB cells), a mouse macrophage cell line (ATCC #TIB-67)in the presence of CpG motifs will contain more IL-6 and TNF-alpha thancells not in the presence of CpG islands. Thus, an IL-6 and TNF-alphasolid phase ELISA was used to quantify the levels of IL-6 and TNF-alphareleased into the supernatants (both from R&D System, Minneapolis,Minn.) upon exposure to various oligonucleotides comprising CpGsequences. For the IL-6 ELISA, a monoclonal antibody specific for mouseIL-6 was pre-coated onto a 96 well microplate. An enzyme-linkedpolyclonal antibody specific for mouse IL-6 was used to detect any boundIL-6 from the cell supernatants. For the TNF-alpha ELISA, an affinitypurified polyclonal antibody specific for mouse TNF-alpha was pre-coatedonto a 96 well microplate. An enzyme-linked polyclonal antibody specificfor mouse TNF-alpha was used to detect any bound TNF-alpha from the cellsupernatants. Both ELISAs used a colorimetric readout quantified byspectrophotometry.

[0321] J774A.1 cells (TIB cells) were cultured in Dulbecco's ModifiedEagle Media (DMEM) with 10% fetal bovine serum (FBS) (Invitrogen,Carlsbad, Calif.) at 37° C., 5% CO₂. 100,000 cells were plated into anappropriate number of wells on a 24 well plate one day prior to theexperiment. The CpG embedded PDGF aptamers were incubated with the cellsfor 24 and 48 hours at 37° C., 5% COY. Final aptamer concentrations were1uM and 10 uM. Supernatants were collected at the indicated time-pointsand centrifuged for 8 minutes at 5,000 rpm at 4° C. Centrifugedsupernatants were frozen at −20° C. until use in the IL-6 or TNF-alphaELISA. Both ELISAs were used according to manufacturer'srecommendations. CpG motif containing oligonucleotides previouslyreported to be immunostimulatory, (ISS ODN and ODN2006) and LPS (Sigma),were used as positive controls and non-CpG containing aptamers were usedas negative controls. FIG. 21A shows the results of an IL-6 ELISAmeasuring IL-6 release in TIB cells using only the standard ODN's aspositive controls, and aptamers which contain no CpG islands as negativecontrols in the assay. Positive and negative controls only were used inthis experiment to establish whether this assay was robust enough tomeasure CpG induced IL-6 release. The data shown in FIG. 21B demonstratethat the ISS ODN and shortened versions of the ISS ODN induce IL-6release in TIB cells better than ODN2006 and shortened versions thereof.FIG. 21C shows the short, long, and full versions of ARC 124 embeddedwith CpG motifs induces IL-6 release in TIB cells as well as the ISSODN. The data shown in FIG. 21D shows that TransARC124.1-TransARC124.7(SEQ ID NO:23-SEQ ID NO:29) induce IL-6 release in TIB cells. The datashown in FIG. 21E shows TransARC124.1-TransARC124.7 induce TNF-alpharelease in TIB cells.

[0322] The results of the IL-6 release and TNF-alpha release assays showthat when CpG motifs are incorporated into existing aptamers, in thiscase ARC124 (SEQ ID NO:11), the aptamer is capable of eliciting a CpGresponse.

Example 13 CpG Island Containing PDGF Aptamers in ERK PhosphorylationAssay

[0323] ERK phosphorylation was used to test whether ARC124 (SEQ IDNO:11) retained its functionality after the incorporation of CpG motifsinto the aptamer sequence. One hundred and fifty thousand 3T3 cells (amouse fibroblast cell line which contains PDGF-R) were plated into anappropriate number of wells on a 12 well plate one day prior to theexperiment and serum starved in 0.5% FBS overnight. Cells were treatedwith 19 ng/ml PDGF-BB (R&D Systems, Minneapolis, Minn.) in the presenceor absence of CpG-island containing PDGF aptamers for 30 minutes. Cellswere collected and lysed with lysis buffer containing 10 mM Tris pH 7.5,100 mM NaCl, 0.125% NP-40, 0.875% Brij 97, 1.5 mM sodium vanadate, and 1mini-EDTA free protease inhibitor tablet. Protein concentration in celllysates was determined using BIO-RAD protein assay reagent according tomanufacturer's recommendations (Bio-Rad, Hercules, Calif.). Lysates wereprepared by adding NuPage LDS Sample Buffer 4× with 0.1M DTT to a finalconcentration of 1× (Invitrogen, Carlsbad, Calif.) and incubated at 70°C. for 7 minutes. Forty micrograms of total protein was loaded into eachwell of a NuPage 10% Bis-Tris gel (Invitrogen, Carlsbad, Calif.). Thegel was run at 100 mAmp for 1 hour. Gel was then transferred to a HyBondECL nitrocellulose membrane (Amersham, Piscataway, N.J.) at 250 mAmp for3 hours. After transfer, the nitrocellulose was blocked with 5% BSA(Sigma, St. Louis, Mo.) in PBS for one hour at room temperature. Thenitrocellulose membrane was incubated with a p44/42 MAP Kinase antibodyovernight at 4° C. (Cell Signaling Technology, Beverly, Mass.). Thenitrocellulose membrane was washed 3× with PBS containing 1% Tween 20and then incubated with an anti-rabbit IgG, HRP-conjugated antibody for1 hour (Amersham, Piscataway, N.J.). After the one hour incubation, thenitrocellulose membrane was washed 3× with PBS containing Tween-20.Membrane was developed using an ECL+Western Blotting Detection Systemaccording to manufacturer's recommendations (Amersham, Piscataway, N.J.)and scanned using a STORM 860 (Amersham). 3T3 cells in the presence ofPDGF-BB without aptamer was used as a positive control.

[0324] The results show that shortARC124 and longARC124, both PDGFaptamers carrying known CpG motifs, are still functionally active andcan block phosphorylation of ERK upon binding to PDGF.

[0325] Collectively, the data show that when CpG motifs are incorporatedinto existing aptamers, the aptamer is capable of eliciting a CpGresponse and still maintain the ability to block non-CpG target (e.g.,PDGF) driven effects (e.g., ERK-MAPK phosphorylation) with the samepotency as native aptamers.

Example 14 PDGF-AA Selection

[0326] PDGF-AA Selection Summary

[0327] One selection for the short form of PDGF-AA (Roche Biomedical)was completed using a 2-fluoro pyrimidine containing pool. Round 1 ofthe selection began with incubation of 2×10¹⁴ molecules of 2° F.pyrimidine modified ARC 212 pool (5′GGGAAAAGCGAAUCAUACACAAGA-N40-GCUCCGCCAGAGACCAACCGAGAA 3′) (SEQ IDNO:91), including a spike of α³²P ATP body labeled pool, with 50 pmolesof PDGF-AA protein in a final volume of 100 μL for 1hr at roomtemperature. The selection was performed in 1× SHMCK buffer, pH 7.4 (20mM Hepes pH 7.4, 120 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂).RNA:PDGF-AA complexes and free RNA molecules were separated using 0.45um nitrocellulose spin columns from Schleicher & Schuell (Keene, N.H.).The column was pre-washed with 1 ml 1×SHMCK, and then theRNA:protein-containing solution was added to the column and spun in acentrifuge at 1500 g for 2 min. Buffer washes were performed to removenonspecific binders from the filters (Round 1, 2×500 μL 1×SHMCK; inlater rounds, more stringent washes (increased number of washes andvolume) were used to enrich for specific binders, then the RNA:proteincomplexes attached to the filters were eluted with 2×200 ul washes(2×100 μL washes in later rounds) of elution buffer (7M urea, 100 mMsodium acetate, 3 mM EDTA, pre-heated to 95° C.). The eluted RNA wasphenol:chloroform extracted, then precipitated (40 μg glycogen, 1 volumeisopropanol). The RNA was reverse transcribed with the ThermoScriptRT-PCR™ system according to their instructions, using the primersKMT.108.38.C (5′ TAATACGACTCACTATAGGGAAAAGCGAATCATACACAAGA 3′) (SEQ IDNO:92) and KMT.108.38.D (5′ TTCTCGGTTGGTCTCTGGCGGAGC 3′) (SEQ ID NO:93),followed by amplification by PCR (20 mM Tris pH 8.4, 50 mM KCl, 2 mMMgCl₂, 0.5 μM KMT.108.38.C, 0.5 μM KMT.108.38.D, 0.5 mM each dNTP, 0.05units/μL Taq polymerase (New England Biolabs)). The PCR template waspurified using the QIAquick PCR purification kit (Qiagen). Templateswere transcribed using α³²P ATP body labeling overnight at 37° C. (4%PEG-8000, 40 mM Tris pH 8.0, 12 mM MgCl₂, 1 mM spermidine, 0.002% Tritonx-100, 3 mM 2′OH purines, 3 mM 2° F. pyrimidines, 25 mM DTT, inorganicpyrophosphatase, T7 Y639F single mutant RNA polymerase, 5 uCi α³²P ATP).The reactions were desalted using Bio Spin columns (Bio-Rad) accordingto the manufacturer's instructions.

[0328] Subsequent rounds were repeated using the same method as forround 1, but with the addition of a negative selection step. Prior toincubation with protein target, the pool RNA was passed through 0.45micron nitrocellulose to remove filter binding sequences, then thefiltrate was carried on into the positive selection step. In alternatingrounds, the pool RNA was gel purified. Transcription reactions werequenched with 50 mM EDTA and ethanol precipitated then purified on a 1.5mm denaturing polyacrylamide gels (8 M urea, 10% acrylamide; 19:1acrylamide:bisacrylamide). Pool RNA was removed from the gel byelectroelution in an Elutrap® apparatus (Schleicher and Schuell, Keene,N.H.) at 225V for 1 hour in 1×TBE (90 mM Tris, 90 mM boric acid, 0.2 mMEDTA). The eluted material was precipitated by the addition of 300 mMsodium acetate and 2.5 volumes of ethanol.

[0329] The RNA remained in excess of the protein throughout theselection (˜1 μM RNA). The protein concentration was 500 nM for thefirst 4 rounds, and then was dropped gradually over the successiverounds. Competitor tRNA was added to the binding reactions at 0.1 mg/mLstarting at Round 2. A total of 11 rounds were completed, with bindingassays performed at select rounds. Table 9 contains the selectiondetails including pool RNA concentration, protein concentration, andtRNA concentration used for each round. Elution values (ratio of CPMvalues of protein-bound RNA versus total RNA flowing through the filtercolumn) along with binding assays were used to monitor selectionprogress. TABLE 9 Conditions used for PDGF-AA (human short form)selection sfh-PDGFAA RNA pool protein tRNA conc protein conc conc PCRRound # (uM) type (uM) (mg/mL) neg % elution cycle # 1 3.3 sfhPDGFAA 0.50 none 0.92 8 2 ˜1 sfhPDGFAA 0.5 0.1 NC 0.24 15 3 ˜1 sfhPDGFAA 0.5 0.1NC 0.46 12 4 ˜1 sfhPDGFAA 0.5 0.1 NC 0.1 15 5 1 sfhPDGFAA 0.4 0.1 NC1.39 10 6 ˜1 sfhPDGFAA 0.4 0.1 NC 0.5 8 7 1 sfhPDGFAA 0.3 0.1 NC 1.23 88 ˜1 sfhPDGFAA 0.3 0.1 NC 0.44 10 9 1 sfhPDGFAA 0.3 0.1 NC 5.05 8 10 ˜1sfhPDGFAA 0.2 0.1 NC 0.83 10 11 1 sfhPDGFAA 0.2 0.1 NC 4.32 7

[0330] Protein Binding Analysis

[0331] Dot blot binding assays were performed throughout the selectionto monitor the protein binding affinity of the pool. Trace ³²P-labeledRNA was combined with PDGF-AA and incubated at room temperature for 30min in 1×SHMCK plus 0.1 mg/ml tRNA for a final volume of 20 μl. Thereaction was added to a dot blot apparatus (Schleicher and SchuellMinifold-1 Dot Blot, Acrylic), assembled (from top to bottom) withnitrocellulose, nylon, and gel blot membranes. RNA that is bound toprotein is captured on the nitrocellulose filter, whereas thenon-protein bound RNA is captured on the nylon filter. When asignificant positive ratio of binding of RNA in the presence of PDGF-AAversus in the absence of PDGF-AA was seen, the pool was cloned using theTOPO TA cloning kit (Invitrogen, Carlsbad, Calif.) according to themanufacturer's instructions. The Round 10 pool template was cloned, and17 sequences were obtained. Only five different sequences were seen,with two major families and one unique sequence (not of the twofamilies). For K_(d) determination, the clone RNA transcripts were 5′endlabeled with γ-³²P ATP. K_(d) values were determined using the dot blotassay and fitting an equation describing a 1:1 RNA:protein complex tothe resulting data (Kaleidagraph, FIGS. 23A and 23B). Results of proteinbinding characterization are tabulated in Table 10. Clones with highaffinity to PDGF-AA were prepped and screened for functionality incell-based assays. TABLE 10 Clone binding activity* R10 PDGF-AA ClonesPDGF-AA (human PDGF-AA short form) (rat) # Clone Name Kd (nM) Kd (nM) 1ARX33P1.D1 N.B. N.B. 2 ARX33P1.D2 104.5 51.7 3 ARX33P1.E5 117.0 57.4 4ARX33P1.E10 132.9 47.1 5 ARX33P1.E11 N.B. N.B.

[0332] PDGF-AA Aptamers

[0333] The sequences for PDGF-AA short form aptamers of the inventionare presented in Table 11. TABLE 11 Sequence Information for PDGF-AA(short form) aptamers ARX33P1.D1 (SEQ ID NO: 94)GGGAAAAGCGAATCATACACAAGATCGCCAGGAGCAAAGTCACGGAGGAGTGGGGGTACGAATGCTCCGCCAGAGACCAACCGAGAA ARX33P1.D2 (SEQ ID NO: 95)GGGAAAAGCGAATCATACACAAGACCGGGAACTCGGATTCTTCGCATGTGGATGCGATCAGTATGCTCCGCCAGAGACCAACCGAGAA ARX33P1.E5 (SEQ ID NO: 96)GGGAAAAGCGAATCATACACAAGACCGGGAACTCGGATTCTTCACATGTGGATGTGATCAGTATGCTCCGCCAGAGACCAACCGAGAA ARX33P1.E10 (SEQ ID NO: 97)GGGAAAAGCGAATCATACACAAGACCGGAAACTCGGATTCTTCGCATGTGGATGCGATCAGTATGCTCCGCCAGAGACCAACCGAGAA ARX33P1.E11 (SEQ ID NO: 98)GGGAAAAGCGAATCATACACAAGAGAGTGGAGGAGGTATGTATGGTTTGTGCGTCTGGTGCGGTGCTCCGCCAGAGACCAACCGAGAA

[0334] Cell Based Assays with PDGF-AA Aptamers

[0335] The PDGF-AA aptamers that showed in vitro binding were tested inthe 3T3 proliferation assay for their ability to inhibit PDGF-AA induced3T3 cell proliferation. The assay was set up as previously described,using a titration of PDGF-AA aptamer (0-1 uM) against a constantconcentration (50 ng/ml) of PDGF-AA protein (R&D Systems). The resultsin FIG. 24 show that the PDGF-AA aptamers ARX33P1.D2, ARX33P1.E5, andARX33P1.E10 do inhibit PDGF-AA induced 3T3 cell proliferation, but arenot very potent. FIG. 25 shows that PDGF-AA aptamers have no effect onPDGF-BB induced 3T3 cell proliferation, indicating that the PDGF-AAaptamers are highly specific for the PDGF-AA isoform.

[0336] The invention having now been described by way of writtendescription and example, those of skill in the art will recognize thatthe invention can be practiced in a variety of embodiments and that thedescription and examples above are for purposes of illustration and notlimitation of the following claims.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 98 <210> SEQ ID NO 1<211> LENGTH: 9 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: aptamer <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (6)..(6) <223> OTHER INFORMATION:2′-Fluoro-Uracil <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (8)..(8) <223> OTHER INFORMATION: 2′-Fluoro-Cytosine <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (9)..(9) <223>OTHER INFORMATION: gm <400> SEQUENCE: 1 caggcuacg 9 <210> SEQ ID NO 2<211> LENGTH: 12 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: aptamer <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (5)..(5) <223> OTHER INFORMATION: gm <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (7)..(7) <223>OTHER INFORMATION: gm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (10)..(10) <223> OTHER INFORMATION: 2′ Fluoro-Uracil <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (11)..(11) <223>OTHER INFORMATION: 2′ Fluoro-Cytosine <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (12)..(12) <223> OTHER INFORMATION:2′-O-methyl-Adenosine <400> SEQUENCE: 2 cgtagagcau ca 12 <210> SEQ ID NO3 <211> LENGTH: 9 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: aptamer <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (5)..(6) <223> OTHER INFORMATION:2′-fluoro-Cytosine <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (7)..(7) <223> OTHER INFORMATION: 2′-fluoro-Uracil <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (8)..(8) <223>OTHER INFORMATION: gm <400> SEQUENCE: 3 tgatccugt 9 <210> SEQ ID NO 4<211> LENGTH: 9 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: aptamer <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (4)..(4) <223> OTHER INFORMATION:2′-fluoro-Cytosine <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (4)..(4) <223> OTHER INFORMATION: 2′-fluoro-cytosine <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (5)..(5) <223>OTHER INFORMATION: gm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (6)..(6) <223> OTHER INFORMATION: 2′-fluoro-Uracil <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (8)..(8) <223>OTHER INFORMATION: 2′-fluoro-Cytosine <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (9)..(9) <223> OTHER INFORMATION: gm <400>SEQUENCE: 4 cagcguacg 9 <210> SEQ ID NO 5 <211> LENGTH: 12 <212> TYPE:DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION:aptamer <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:(7)..(7) <223> OTHER INFORMATION: gm <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (10)..(10) <223> OTHER INFORMATION:2′-fluoro-Uracil <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (11)..(11) <223> OTHER INFORMATION: 2′-fluoro-Cytosine <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (12)..(12) <223>OTHER INFORMATION: 2′-methyl-Adenosine <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (12)..(12) <223> OTHER INFORMATION:2′-O-methyl-adenosine <400> SEQUENCE: 5 cgtaccgatu ca 12 <210> SEQ ID NO6 <211> LENGTH: 9 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: aptamer <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (6)..(6) <223> OTHER INFORMATION:2′-fluoro-Cytosine <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (7)..(7) <223> OTHER INFORMATION: 2′-fluoro-Uracil <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (8)..(8) <223>OTHER INFORMATION: gm <400> SEQUENCE: 6 tgaagcugt 9 <210> SEQ ID NO 7<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: aptamer <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (1)..(1) <223> OTHER INFORMATION:2′-O-methyl-adenosine <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (2)..(2) <223> OTHER INFORMATION: 2′-O-methyl-uracil <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (3)..(3) <223>OTHER INFORMATION: gm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (4)..(4) <223> OTHER INFORMATION: cm <220> FEATURE: <221>NAME/KEY: modified_base <222> LOCATION: (5)..(5) <223> OTHERINFORMATION: 2′-O-methyl-adenosine <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (6)..(6) <223> OTHER INFORMATION: gm <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (7)..(9) <223>OTHER INFORMATION: 2′-O-methyl-uracil <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (10)..(10) <223> OTHER INFORMATION: gm<220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (11)..(11)<223> OTHER INFORMATION: 2′-O-methyl-adenosine <220> FEATURE: <221>NAME/KEY: modified_base <222> LOCATION: (12)..(12) <223> OTHERINFORMATION: gm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (13)..(14) <223> OTHER INFORMATION: 2′-O-methyl-adenosine<220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (15)..(15)<223> OTHER INFORMATION: gm <220> FEATURE: <221> NAME/KEY: modified_base<222> LOCATION: (16)..(16) <223> OTHER INFORMATION: 2′-O-methyl-uracil<220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (17)..(17)<223> OTHER INFORMATION: cm <220> FEATURE: <221> NAME/KEY: modified_base<222> LOCATION: (18)..(18) <223> OTHER INFORMATION: gm <220> FEATURE:<221> NAME/KEY: modified_base <222> LOCATION: (19)..(19) <223> OTHERINFORMATION: cm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (20)..(20) <223> OTHER INFORMATION: gm <220> FEATURE: <221>NAME/KEY: modified_base <222> LOCATION: (21)..(21) <223> OTHERINFORMATION: cm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (22)..(22) <223> OTHER INFORMATION: 2′-O-methyl-adenosine<220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (23)..(23)<223> OTHER INFORMATION: 2′-O-methyl-uracil <400> SEQUENCE: 7 augcaguuugagaagucgcg cau 23 <210> SEQ ID NO 8 <211> LENGTH: 29 <212> TYPE: DNA<213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION:aptamer <400> SEQUENCE: 8 caggctacgc gtagagcatc atgatcctg 29 <210> SEQID NO 9 <211> LENGTH: 56 <212> TYPE: DNA <213> ORGANISM: Artificial<220> FEATURE: <223> OTHER INFORMATION: aptamer <220> FEATURE: <221>NAME/KEY: modified_base <222> LOCATION: (10)..(10) <223> OTHERINFORMATION: 2′-O-methyl-adenosine <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (11)..(11) <223> OTHER INFORMATION:2′-O-methyl-uracil <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (12)..(12) <223> OTHER INFORMATION: gm <220> FEATURE: <221>NAME/KEY: modified_base <222> LOCATION: (13)..(13) <223> OTHERINFORMATION: cm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (14)..(14) <223> OTHER INFORMATION: 2′-O-methyl-adenosine<220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (15)..(15)<223> OTHER INFORMATION: gm <220> FEATURE: <221> NAME/KEY: modified_base<222> LOCATION: (16)..(18) <223> OTHER INFORMATION: 2′-O-methyl-uracil<220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (19)..(19)<223> OTHER INFORMATION: gm <220> FEATURE: <221> NAME/KEY: modified_base<222> LOCATION: (20)..(20) <223> OTHER INFORMATION:2′-O-methyl-adenosine <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (21)..(21) <223> OTHER INFORMATION: gm <220> FEATURE: <221>NAME/KEY: modified_base <222> LOCATION: (22)..(23) <223> OTHERINFORMATION: 2′-O-methyl-adenosine <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (24)..(24) <223> OTHER INFORMATION: gm<220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (25)..(25)<223> OTHER INFORMATION: 2′-O-methyl-uracil <220> FEATURE: <221>NAME/KEY: modified_base <222> LOCATION: (26)..(26) <223> OTHERINFORMATION: cm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (27)..(27) <223> OTHER INFORMATION: gm <220> FEATURE: <221>NAME/KEY: modified_base <222> LOCATION: (28)..(28) <223> OTHERINFORMATION: cm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (29)..(29) <223> OTHER INFORMATION: gm <220> FEATURE: <221>NAME/KEY: modified_base <222> LOCATION: (30)..(30) <223> OTHERINFORMATION: cm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (31)..(31) <223> OTHER INFORMATION: 2′-O-methyl-adenosine<220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (32)..(32)<223> OTHER INFORMATION: 2′-O-methyl-uracil <400> SEQUENCE: 9 caggctacgaugcaguuuga gaagucgcgc aucgtagagc atcagaaatg atcctg 56 <210> SEQ ID NO 10<211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: aptamer <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (10)..(10) <223> OTHER INFORMATION:2′-O-methyl-uracil <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (11)..(11) <223> OTHER INFORMATION: gm <220> FEATURE: <221>NAME/KEY: modified_base <222> LOCATION: (12)..(12) <223> OTHERINFORMATION: cm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (13)..(13) <223> OTHER INFORMATION: 2′-O-methyl-adenosine<220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (14)..(14)<223> OTHER INFORMATION: gm <220> FEATURE: <221> NAME/KEY: modified_base<222> LOCATION: (15)..(17) <223> OTHER INFORMATION: 2′-O-methyl-uracil<220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (18)..(18)<223> OTHER INFORMATION: gm <220> FEATURE: <221> NAME/KEY: modified_base<222> LOCATION: (19)..(19) <223> OTHER INFORMATION:2′-O-methyl-adenosine <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (20)..(20) <223> OTHER INFORMATION: gm <220> FEATURE: <221>NAME/KEY: modified_base <222> LOCATION: (21)..(22) <223> OTHERINFORMATION: 2′-O-methyl-adenosine <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (23)..(23) <223> OTHER INFORMATION: gm<220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (24)..(24)<223> OTHER INFORMATION: 2′-O-methyl-uracil <220> FEATURE: <221>NAME/KEY: modified_base <222> LOCATION: (25)..(25) <223> OTHERINFORMATION: cm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (26)..(26) <223> OTHER INFORMATION: gm <220> FEATURE: <221>NAME/KEY: modified_base <222> LOCATION: (27)..(27) <223> OTHERINFORMATION: cm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (28)..(28) <223> OTHER INFORMATION: gm <220> FEATURE: <221>NAME/KEY: modified_base <222> LOCATION: (29)..(29) <223> OTHERINFORMATION: cm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (30)..(30) <223> OTHER INFORMATION: 2′-O-methyl-adenosine<400> SEQUENCE: 10 caggctacgu gcaguuugag aagucgcgca cgtagagcatcagaaatgat cctg 54 <210> SEQ ID NO 11 <211> LENGTH: 39 <212> TYPE: DNA<213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION:aptamer <400> SEQUENCE: 11 cacaggctac ggcacgtaga gcatcaccat gatcctgtg 39<210> SEQ ID NO 12 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM:Artificial <220> FEATURE: <223> OTHER INFORMATION: aptamer <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(22) <223>OTHER INFORMATION: phosphorothioate backbone <400> SEQUENCE: 12tgactgtgaa cgttcgagat ga 22 <210> SEQ ID NO 13 <211> LENGTH: 14 <212>TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: aptamer <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (1)..(14) <223> OTHER INFORMATION: phosphorothioate backbone<400> SEQUENCE: 13 tgaacgttcg agat 14 <210> SEQ ID NO 14 <211> LENGTH:12 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: aptamer <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (1)..(12) <223> OTHER INFORMATION: phosphorothioate backbone<400> SEQUENCE: 14 aacgttcgag at 12 <210> SEQ ID NO 15 <211> LENGTH: 10<212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: aptamer <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (1)..(10) <223> OTHER INFORMATION: phosphorothioate backbone<400> SEQUENCE: 15 aacgttcgag 10 <210> SEQ ID NO 16 <211> LENGTH: 13<212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: aptamer <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (1)..(13) <223> OTHER INFORMATION: phosphorothioate backbone<400> SEQUENCE: 16 gtgaacgttc gag 13 <210> SEQ ID NO 17 <211> LENGTH: 24<212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: aptamer <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (1)..(24) <223> OTHER INFORMATION: phosphorothioate backbone<400> SEQUENCE: 17 tcgtcgtttt gtcgttttgt cgtt 24 <210> SEQ ID NO 18<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: aptamer <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (1)..(18) <223> OTHER INFORMATION:phosphorothioate backbone <400> SEQUENCE: 18 gtcgttttgt cgttttgt 18<210> SEQ ID NO 19 <211> LENGTH: 14 <212> TYPE: DNA <213> ORGANISM:Artificial <220> FEATURE: <223> OTHER INFORMATION: aptamer <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(14) <223>OTHER INFORMATION: phosphorothioate backbone <400> SEQUENCE: 19gtcgttttgt cgtt 14 <210> SEQ ID NO 20 <211> LENGTH: 46 <212> TYPE: DNA<213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION:aptamer <400> SEQUENCE: 20 aacgttcgag caggctacgg cacgtagagc atcaccatgatcctgc 46 <210> SEQ ID NO 21 <211> LENGTH: 49 <212> TYPE: DNA <213>ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: aptamer<400> SEQUENCE: 21 gtgaacgttc gagcaggcta cggcacgtag agcatcacca tgatcctgc49 <210> SEQ ID NO 22 <211> LENGTH: 64 <212> TYPE: DNA <213> ORGANISM:Artificial <220> FEATURE: <223> OTHER INFORMATION: aptamer <400>SEQUENCE: 22 tgactgtgaa cgttcgagat gacaggctac ggcacgtaga gcatcaccatgatcctgttt 60 tttt 64 <210> SEQ ID NO 23 <211> LENGTH: 34 <212> TYPE:DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION:aptamer <400> SEQUENCE: 23 caggctacgt tcgtagagca tcaccatgat cctg 34<210> SEQ ID NO 24 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM:Artificial <220> FEATURE: <223> OTHER INFORMATION: aptamer <400>SEQUENCE: 24 caggctacgt ttcgtagagc atcaccatga tcctg 35 <210> SEQ ID NO25 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: aptamer <400> SEQUENCE: 25 caggcaacgtttcgttgagc atcaccatga tcctg 35 <210> SEQ ID NO 26 <211> LENGTH: 34 <212>TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: aptamer <400> SEQUENCE: 26 caggcaacgt tcgttgagca tcaccatgatcctg 34 <210> SEQ ID NO 27 <211> LENGTH: 36 <212> TYPE: DNA <213>ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: aptamer<400> SEQUENCE: 27 caggcaacgt tttcgttgag catcaccatg atcctg 36 <210> SEQID NO 28 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial<220> FEATURE: <223> OTHER INFORMATION: aptamer <400> SEQUENCE: 28caggctacgt ttcgtagagc atcaccatga tcctg 35 <210> SEQ ID NO 29 <211>LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE:<223> OTHER INFORMATION: aptamer <400> SEQUENCE: 29 caggctacgtttcgtagagc atcaccatga tcctg 35 <210> SEQ ID NO 30 <211> LENGTH: 36 <212>TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: aptamer <400> SEQUENCE: 30 caggcgtcgt tttcgacgag catcaccatgatcctg 36 <210> SEQ ID NO 31 <211> LENGTH: 36 <212> TYPE: DNA <213>ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: aptamer<400> SEQUENCE: 31 caggcgtcgt cgtcgacgag catcaccatg atcctg 36 <210> SEQID NO 32 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial<220> FEATURE: <223> OTHER INFORMATION: aptamer <400> SEQUENCE: 32caggcttcgt cgtcgaagag catcaccatg atcctg 36 <210> SEQ ID NO 33 <211>LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE:<223> OTHER INFORMATION: aptamer <400> SEQUENCE: 33 caggctacgtcgtcgtagag catcaccatg atcctg 36 <210> SEQ ID NO 34 <211> LENGTH: 35<212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: aptamer <400> SEQUENCE: 34 caggcaagct ttgcttgagc atcaccatgatcctg 35 <210> SEQ ID NO 35 <211> LENGTH: 36 <212> TYPE: DNA <213>ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: aptamer<400> SEQUENCE: 35 caggcaagct tttgcttgag catcaccatg atcctg 36 <210> SEQID NO 36 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial<220> FEATURE: <223> OTHER INFORMATION: aptamer <400> SEQUENCE: 36cacaggctac ggcacgtaga gcatcaccat gat 33 <210> SEQ ID NO 37 <211> LENGTH:10 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: aptamer <400> SEQUENCE: 37 aacgttcgag 10 <210> SEQ ID NO 38<400> SEQUENCE: 38 000 <210> SEQ ID NO 39 <400> SEQUENCE: 39 000 <210>SEQ ID NO 40 <400> SEQUENCE: 40 000 <210> SEQ ID NO 41 <400> SEQUENCE:41 000 <210> SEQ ID NO 42 <400> SEQUENCE: 42 000 <210> SEQ ID NO 43<400> SEQUENCE: 43 000 <210> SEQ ID NO 44 <400> SEQUENCE: 44 000 <210>SEQ ID NO 45 <400> SEQUENCE: 45 000 <210> SEQ ID NO 46 <400> SEQUENCE:46 000 <210> SEQ ID NO 47 <400> SEQUENCE: 47 000 <210> SEQ ID NO 48<400> SEQUENCE: 48 000 <210> SEQ ID NO 49 <400> SEQUENCE: 49 000 <210>SEQ ID NO 50 <211> LENGTH: 93 <212> TYPE: DNA <213> ORGANISM: Artificial<220> FEATURE: <223> OTHER INFORMATION: pool <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (25)..(54) <223> OTHERINFORMATION: n is a, c, g, t or u <400> SEQUENCE: 50 catcgatgctagtcgtaacg atccnnnnnn nnnnnnnnnn nnnnnnnnnn nnnncgagaa 60 cgttctctcctctccctata gtgagtcgta tta 93 <210> SEQ ID NO 51 <211> LENGTH: 92 <212>TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: pool <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (24)..(53) <223> OTHER INFORMATION: n is a, c, g, t or u <400>SEQUENCE: 51 catgcatcgc gactgactag ccgnnnnnnn nnnnnnnnnn nnnnnnnnnnnnngtagaac 60 gttctctcct ctccctatag tgagtcgtat ta 92 <210> SEQ ID NO 52<211> LENGTH: 92 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: pool <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (24)..(53) <223> OTHER INFORMATION: n is a,c, g, t or u <400> SEQUENCE: 52 catcgatcga tcgatcgaca gcgnnnnnnnnnnnnnnnnn nnnnnnnnnn nnngtagaac 60 gttctctcct ctccctatag tgagtcgtat ta92 <210> SEQ ID NO 53 <211> LENGTH: 9 <212> TYPE: DNA <213> ORGANISM:Artificial <220> FEATURE: <223> OTHER INFORMATION: aptamer <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (9)..(9) <223>OTHER INFORMATION: gm <400> SEQUENCE: 53 caggctacg 9 <210> SEQ ID NO 54<211> LENGTH: 12 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: aptamer <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (5)..(5) <223> OTHER INFORMATION: gm <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (7)..(7) <223>OTHER INFORMATION: gm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (12)..(12) <223> OTHER INFORMATION: 2′-O-methyl-adenosine<400> SEQUENCE: 54 cgtagagcat ca 12 <210> SEQ ID NO 55 <211> LENGTH: 8<212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: aptamer <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (8)..(8) <223> OTHER INFORMATION: gm <400> SEQUENCE: 55tgatcctg 8 <210> SEQ ID NO 56 <211> LENGTH: 9 <212> TYPE: DNA <213>ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: aptamer<220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (6)..(6)<223> OTHER INFORMATION: 2′-O-methyl-uracil <220> FEATURE: <221>NAME/KEY: modified_base <222> LOCATION: (8)..(8) <223> OTHERINFORMATION: cm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (9)..(9) <223> OTHER INFORMATION: gm <400> SEQUENCE: 56caggcuacg 9 <210> SEQ ID NO 57 <211> LENGTH: 12 <212> TYPE: DNA <213>ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: aptamer<220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (5)..(5)<223> OTHER INFORMATION: gm <220> FEATURE: <221> NAME/KEY: modified_base<222> LOCATION: (7)..(7) <223> OTHER INFORMATION: gm <220> FEATURE:<221> NAME/KEY: modified_base <222> LOCATION: (10)..(10) <223> OTHERINFORMATION: 2′-O-methyl-uracil <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (11)..(11) <223> OTHER INFORMATION: cm<220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (12)..(12)<223> OTHER INFORMATION: 2′-O-methyl-adenosine <400> SEQUENCE: 57cgtagagcau ca 12 <210> SEQ ID NO 58 <211> LENGTH: 8 <212> TYPE: DNA<213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION:aptamer <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:(5)..(6) <223> OTHER INFORMATION: cm <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (7)..(7) <223> OTHER INFORMATION:2′-O-methyl-uracil <400> SEQUENCE: 58 tgatccug 8 <210> SEQ ID NO 59<211> LENGTH: 9 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: aptamer <400> SEQUENCE: 59 caggctacg 9<210> SEQ ID NO 60 <211> LENGTH: 12 <212> TYPE: DNA <213> ORGANISM:Artificial <220> FEATURE: <223> OTHER INFORMATION: aptamer <400>SEQUENCE: 60 cgtagagcat ca 12 <210> SEQ ID NO 61 <211> LENGTH: 8 <212>TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: aptamer <400> SEQUENCE: 61 tgatcctg 8 <210> SEQ ID NO 62<211> LENGTH: 9 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: aptamer <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (1)..(1) <223> OTHER INFORMATION: cm <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (2)..(2) <223>OTHER INFORMATION: 2′-O-methyl-adenosine <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (3)..(4) <223> OTHER INFORMATION: gm <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (5)..(5) <223>OTHER INFORMATION: cm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (6)..(6) <223> OTHER INFORMATION: 2′-O-methyl-uracil <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (7)..(7) <223>OTHER INFORMATION: 2′-O-methyl-adenosine <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (8)..(8) <223> OTHER INFORMATION: cm <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (9)..(9) <223>OTHER INFORMATION: gm <400> SEQUENCE: 62 caggcuacg 9 <210> SEQ ID NO 63<211> LENGTH: 12 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: aptamer <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (1)..(1) <223> OTHER INFORMATION: cm <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (2)..(2) <223>OTHER INFORMATION: gm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (3)..(3) <223> OTHER INFORMATION: 2′-O-methyl-uracil <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (4)..(4) <223>OTHER INFORMATION: 2′-O-methyl-adenosine <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (5)..(5) <223> OTHER INFORMATION: gm <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (6)..(6) <223>OTHER INFORMATION: 2′-O-methyl-adenosine <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (7)..(7) <223> OTHER INFORMATION: gm <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (8)..(8) <223>OTHER INFORMATION: cm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (9)..(9) <223> OTHER INFORMATION: 2′-O-methyl-adenosine <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (10)..(10) <223>OTHER INFORMATION: 2′-O-methyl-uracil <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (11)..(11) <223> OTHER INFORMATION: cm<220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (12)..(12)<223> OTHER INFORMATION: 2′-O-methyl-adenosine <400> SEQUENCE: 63cguagagcau ca 12 <210> SEQ ID NO 64 <211> LENGTH: 8 <212> TYPE: DNA<213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION:aptamer <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:(1)..(1) <223> OTHER INFORMATION: 2′-O-methyl-uracil <220> FEATURE:<221> NAME/KEY: modified_base <222> LOCATION: (2)..(2) <223> OTHERINFORMATION: gm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (3)..(3) <223> OTHER INFORMATION: 2′-O-methyl-adenosine <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (4)..(4) <223>OTHER INFORMATION: 2′-O-methyl-uracil <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (5)..(6) <223> OTHER INFORMATION: cm <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (7)..(7) <223>OTHER INFORMATION: 2′-O-methyl-uracil <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (8)..(8) <223> OTHER INFORMATION: gm <400>SEQUENCE: 64 ugauccug 8 <210> SEQ ID NO 65 <211> LENGTH: 10 <212> TYPE:DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION:aptamer <400> SEQUENCE: 65 acaggctacg 10 <210> SEQ ID NO 66 <211>LENGTH: 9 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE:<223> OTHER INFORMATION: aptamer <400> SEQUENCE: 66 tgatcctgt 9 <210>SEQ ID NO 67 <211> LENGTH: 11 <212> TYPE: DNA <213> ORGANISM: Artificial<220> FEATURE: <223> OTHER INFORMATION: aptamer <400> SEQUENCE: 67cacaggctac g 11 <210> SEQ ID NO 68 <211> LENGTH: 10 <212> TYPE: DNA<213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION:aptamer <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:(7)..(7) <223> OTHER INFORMATION: 2′-O-methyl-uracil <400> SEQUENCE: 68tgatccugtg 10 <210> SEQ ID NO 69 <211> LENGTH: 8 <212> TYPE: DNA <213>ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: aptamer<220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (5)..(6)<223> OTHER INFORMATION: cm <220> FEATURE: <221> NAME/KEY: modified_base<222> LOCATION: (8)..(8) <223> OTHER INFORMATION: gm <400> SEQUENCE: 69tgatcctg 8 <210> SEQ ID NO 70 <211> LENGTH: 9 <212> TYPE: DNA <213>ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: aptamer<220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(1)<223> OTHER INFORMATION: cm <220> FEATURE: <221> NAME/KEY: modified_base<222> LOCATION: (2)..(2) <223> OTHER INFORMATION: 2′-O-methyl-adenosine<220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (3)..(4)<223> OTHER INFORMATION: gm <400> SEQUENCE: 70 caggctacg 9 <210> SEQ IDNO 71 <211> LENGTH: 9 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: aptamer <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (9)..(9) <223> OTHER INFORMATION:2′-O-methyl-uracil <400> SEQUENCE: 71 tgatcctgu 9 <210> SEQ ID NO 72<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: aptamer <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (5)..(6) <223> OTHER INFORMATION: cm <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (7)..(7) <223>OTHER INFORMATION: 2′-O-methyl-uracil <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (8)..(8) <223> OTHER INFORMATION: gm <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (9)..(9) <223>OTHER INFORMATION: 2′-O-methyl-uracil <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (10)..(10) <223> OTHER INFORMATION: gm<400> SEQUENCE: 72 tgatccugug 10 <210> SEQ ID NO 73 <211> LENGTH: 9<212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: aptamer <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (8)..(8) <223> OTHER INFORMATION: cm <220> FEATURE: <221>NAME/KEY: modified_base <222> LOCATION: (9)..(9) <223> OTHERINFORMATION: gm <400> SEQUENCE: 73 caggctacg 9 <210> SEQ ID NO 74 <211>LENGTH: 9 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE:<223> OTHER INFORMATION: aptamer <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (6)..(6) <223> OTHER INFORMATION:2′-O-methyl-uracil <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (9)..(9) <223> OTHER INFORMATION: gm <400> SEQUENCE: 74caggcuacg 9 <210> SEQ ID NO 75 <211> LENGTH: 12 <212> TYPE: DNA <213>ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: aptamer<220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (5)..(5)<223> OTHER INFORMATION: gm <220> FEATURE: <221> NAME/KEY: modified_base<222> LOCATION: (7)..(7) <223> OTHER INFORMATION: gm <220> FEATURE:<221> NAME/KEY: modified_base <222> LOCATION: (11)..(11) <223> OTHERINFORMATION: cm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (12)..(12) <223> OTHER INFORMATION: 2′-O-methyl-adenosine<400> SEQUENCE: 75 cgtagagcat ca 12 <210> SEQ ID NO 76 <211> LENGTH: 12<212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: aptamer <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (5)..(5) <223> OTHER INFORMATION: gm <220> FEATURE: <221>NAME/KEY: modified_base <222> LOCATION: (7)..(7) <223> OTHERINFORMATION: gm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (10)..(10) <223> OTHER INFORMATION: 2′-O-methyl-uracil <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (12)..(12) <223>OTHER INFORMATION: 2′-O-methyl-adenosine <400> SEQUENCE: 76 cgtagagcauca 12 <210> SEQ ID NO 77 <211> LENGTH: 9 <212> TYPE: DNA <213> ORGANISM:Artificial <220> FEATURE: <223> OTHER INFORMATION: aptamer <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (5)..(5) <223>OTHER INFORMATION: cm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (6)..(6) <223> OTHER INFORMATION: 2′-O-methyl-uracil <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (8)..(8) <223>OTHER INFORMATION: cm <400> SEQUENCE: 77 caggcuacg 9 <210> SEQ ID NO 78<211> LENGTH: 12 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: aptamer <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (1)..(1) <223> OTHER INFORMATION: cm <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (3)..(3) <223>OTHER INFORMATION: 2′-O-methyl-uracil <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (8)..(8) <223> OTHER INFORMATION: cm <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (10)..(10) <223>OTHER INFORMATION: 2′-O-methyl-uracil <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (11)..(11) <223> OTHER INFORMATION: cm<400> SEQUENCE: 78 cguagagcau ca 12 <210> SEQ ID NO 79 <211> LENGTH: 8<212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: aptamer <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (1)..(1) <223> OTHER INFORMATION: 2′-O-methyl-uracil <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (4)..(4) <223>OTHER INFORMATION: 2′-O-methyl-uracil <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (5)..(6) <223> OTHER INFORMATION: cm <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (7)..(7) <223>OTHER INFORMATION: 2′-O-methyl-uracil <400> SEQUENCE: 79 ugauccug 8<210> SEQ ID NO 80 <211> LENGTH: 8 <212> TYPE: DNA <213> ORGANISM:Artificial <220> FEATURE: <223> OTHER INFORMATION: aptamer <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (6)..(6) <223>OTHER INFORMATION: cm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (7)..(7) <223> OTHER INFORMATION: 2′-O-methyl-uracil <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (8)..(8) <223>OTHER INFORMATION: gm <400> SEQUENCE: 80 tgatccug 8 <210> SEQ ID NO 81<211> LENGTH: 11 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: aptamer <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (11)..(11) <223> OTHER INFORMATION: gm<400> SEQUENCE: 81 cacaggctac g 11 <210> SEQ ID NO 82 <211> LENGTH: 10<212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: aptamer <400> SEQUENCE: 82 tgatcctgtg 10 <210> SEQ ID NO 83<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: aptamer <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (6)..(6) <223> OTHER INFORMATION: cm <220>FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (7)..(7) <223>OTHER INFORMATION: 2′-O-methyl-uracil <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (9)..(9) <223> OTHER INFORMATION:2′-O-methyl-uracil <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (10)..(10) <223> OTHER INFORMATION: gm <400> SEQUENCE: 83tgatccugug 10 <210> SEQ ID NO 84 <211> LENGTH: 11 <212> TYPE: DNA <213>ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: aptamer<220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (8)..(8)<223> OTHER INFORMATION: 2′-O-methyl-uracil <220> FEATURE: <221>NAME/KEY: modified_base <222> LOCATION: (10)..(10) <223> OTHERINFORMATION: cm <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (11)..(11) <223> OTHER INFORMATION: gm <400> SEQUENCE: 84cacaggcuac g 11 <210> SEQ ID NO 85 <211> LENGTH: 8 <212> TYPE: DNA <213>ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: aptamer<220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (5)..(5)<223> OTHER INFORMATION: cm <220> FEATURE: <221> NAME/KEY: modified_base<222> LOCATION: (7)..(7) <223> OTHER INFORMATION: 2′-O-methyl-uracil<220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (8)..(8)<223> OTHER INFORMATION: gm <400> SEQUENCE: 85 tgatccug 8 <210> SEQ IDNO 86 <211> LENGTH: 11 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: aptamer <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (11)..(11) <223> OTHER INFORMATION: gm<400> SEQUENCE: 86 cccaggctac g 11 <210> SEQ ID NO 87 <211> LENGTH: 10<212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: aptamer <220> FEATURE: <221> NAME/KEY: modified_base <222>LOCATION: (5)..(5) <223> OTHER INFORMATION: cm <220> FEATURE: <221>NAME/KEY: modified_base <222> LOCATION: (8)..(10) <223> OTHERINFORMATION: gm <400> SEQUENCE: 87 tgatcctggg 10 <210> SEQ ID NO 88<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: aptamer <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (8)..(10) <223> OTHER INFORMATION: gm<400> SEQUENCE: 88 tgatcctggg 10 <210> SEQ ID NO 89 <211> LENGTH: 10<212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: aptamer <400> SEQUENCE: 89 tgatcctggg 10 <210> SEQ ID NO 90<211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: aptamer <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (5)..(5) <223> OTHER INFORMATION: cm <400>SEQUENCE: 90 tgatcctggg 10 <210> SEQ ID NO 91 <211> LENGTH: 88 <212>TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: pool <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (25)..(64) <223> OTHER INFORMATION: n is a, c, g, t or u <400>SEQUENCE: 91 gggaaaagcg aaucauacac aagannnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn 60 nnnngcuccg ccagagacca accgagaa 88 <210> SEQ ID NO 92 <211>LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE:<223> OTHER INFORMATION: aptamer <400> SEQUENCE: 92 taatacgactcactataggg aaaagcgaat catacacaag a 41 <210> SEQ ID NO 93 <211> LENGTH:24 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: aptamer <400> SEQUENCE: 93 ttctcggttg gtctctggcg gagc 24<210> SEQ ID NO 94 <211> LENGTH: 87 <212> TYPE: DNA <213> ORGANISM:Artificial <220> FEATURE: <223> OTHER INFORMATION: aptamer <400>SEQUENCE: 94 gggaaaagcg aatcatacac aagatcgcca ggagcaaagt cacggaggagtgggggtacg 60 aatgctccgc cagagaccaa ccgagaa 87 <210> SEQ ID NO 95 <211>LENGTH: 88 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE:<223> OTHER INFORMATION: aptamer <400> SEQUENCE: 95 gggaaaagcgaatcatacac aagaccggga actcggattc ttcgcatgtg gatgcgatca 60 gtatgctccgccagagacca accgagaa 88 <210> SEQ ID NO 96 <211> LENGTH: 88 <212> TYPE:DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION:aptamer <400> SEQUENCE: 96 gggaaaagcg aatcatacac aagaccggga actcggattcttcacatgtg gatgtgatca 60 gtatgctccg ccagagacca accgagaa 88 <210> SEQ IDNO 97 <211> LENGTH: 88 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: aptamer <400> SEQUENCE: 97 gggaaaagcgaatcatacac aagaccggaa actcggattc ttcgcatgtg gatgcgatca 60 gtatgctccgccagagacca accgagaa 88 <210> SEQ ID NO 98 <211> LENGTH: 88 <212> TYPE:DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION:aptamer <400> SEQUENCE: 98 gggaaaagcg aatcatacac aagagagtgg aggaggtatgtatggtttgt gcgtctggtg 60 cggtgctccg ccagagacca accgagaa 88

What is claimed is: 1) An aptamer that binds to PDGF comprising asequence selected from the group consisting of SEQ ID NO:1 to SEQ IDNO:3, SEQ ID NO:9 to SEQ ID NO:38, SEQ ID NO:50, SEQ ID NO:54 to SEQ IDNO:90, and SEQ ID NO:94 to SEQ ID NO:99. 2) An aptamer that binds toPDGF comprising a sequence containing less than seven nucleotides havinga 2′ fluoro substituent. 3) An aptamer comprising a first sequencecapable of binding to a first target and a second sequence capable ofbinding to a second target. 4) The aptamer of claim 3, wherein the firsttarget is selected from the group consisting of PDGF, PDGF-isoforms, andPDGF receptor and the second target is selected from the groupconsisting of VEGF and VEGF receptor. 5) The aptamer of claim 4,comprising a sequence selected from the group consisting of SEQ ID NO:1to SEQ ID NO:3, SEQ ID NO:9 to SEQ ID NO:38, SEQ ID NO:50, SEQ ID NO:54to SEQ ID NO:90, and SEQ ID NO:94 to SEQ ID NO:99. 6) The aptamer ofclaim 4, wherein the PDGF isoforms are PDGF AA, PDGF BB, PDGF AB, PDGFCC, and PDGF DD. 7) The aptamer of claim 3, wherein said first targetdoes not upon binding of the aptamer stimulate an immune response andfurther wherein said second target does upon binding of the aptamerstimulate an immune response. 8) The aptamer of claim 7, wherein saidsecond target is selected from the group consisting of toll-likereceptors. 9) The aptamer of claim 3, wherein said second sequence is animmunostimulatory sequence. 10) The aptamer of claim 9, wherein theimmunostimulatory sequence is a CpG motif. 11) The aptamer of claim 9,wherein the first sequence is capable of binding to a target selectedfrom the group consisting of PDGF, IgE, IgE Fcε R1, PSMA, CD22,TNF-alpha, CTLA4, PD-1, PD-L1, PD-L2, FcRIIB, BTLA, TIM-3, CD11c, BAFF,B7-X, CD19, CD20, CD25, and CD33. 12) The aptamer of claim 9, whereinthe first sequence is capable of binding to PDGF. 13) A compositioncomprising an aptamer according to any of claims 1 through 12 and apharmaceutically acceptable carrier. 14) A composition comprising anaptamer according to any of claims 1 through 12, a cytotoxic agent and apharmaceutically acceptable carrier. 15) The composition of claim 14,wherein the cytotoxic agent belongs to a class of cytotoxic agentsselected from the group consisting of tubulin stabilizers, tubulindestabilizers, anti-metabolites, purine synthesis inhibitors, nucleosideanalogs, DNA alkylating agents, DNA modifying agents, and vasculardisrupting agents. 16) The composition of claim 14, wherein thecytotoxic agent is used alone or in combinations of one or morecytotoxic agents selected from the group consisting of calicheamycin,doxorubicin, taxol, methotrexate, gemcitabine, cytarabine, vinblastin,daunorubicin, docetaxel, irinotecan, epothilone B, epothilone D,cisplatin, carboplatin, and 5-fluoro-U. 17) A composition comprising anaptamer according to claim 1, an aptamer that binds to VEGF and apharmaceutically acceptable carrier. 18) The composition of claim 17further comprising a cytotoxic agent. 19) The composition of claim 18,wherein the cytotoxic agent belongs to a class of cytotoxic agentsselected from the group consisting of tubulin stabilizers, tubulindestabilizers, anti-metabolites, purine synthesis inhibitors, nucleosideanalogs, DNA alkylating agents, DNA modifying agents, and vasculardisrupting agents. 20) The composition of claim 18, wherein thecytotoxic agent is used alone or in combinations of one or morecytotoxic agents selected from the group consisting of calicheamycin,doxorubicin, taxol, methotrexate, gemcitabine, cytarabine, vinblastin,daunorubicin, docetaxel, irinotecan, epothilone B, epothilone D,cisplatin, carboplatin, and 5-fluoro-U. 21) A method of treating cancercomprising the step of administering a therapeutically effective amountof an aptamer according to any of claims 1 through
 12. 22) A method oftreating cancer comprising the step of administering a therapeuticallyeffective amount of a composition according to any of claims 13, 14 and18. 23) The method of claim 22, wherein the cytotoxic agent belongs to aclass of cytotoxic agents selected from the group consisting of tubulinstabilizers, tubulin destabilizers, anti-metabolites, purine synthesisinhibitors, nucleoside analogs, DNA alkylating agents, DNA modifyingagents, and vascular disrupting agents. 24) The method of claim 22,wherein the cytotoxic agent is used alone or in combinations of one ormore cytotoxic agents selected from the group consisting ofcalicheamycin, doxorubicin, taxol, methotrexate, gemcitabine,cytarabine, vinblastin, daunorubicin, docetaxel, irinotecan, epothiloneB, epothilone D, cisplatin, carboplatin, and 5-fluoro-U. 25) A method ofinhibiting growth of a tumor comprising the step of administering atherapeutically effective amount of an aptamer according to any ofclaims 1 through
 12. 26) A method of inhibiting growth of a tumorcomprising the step of administering a therapeutically effective amountof a composition according to any of claims 13, 14 and
 18. 27) Themethod of claim 26, wherein the cytotoxic agent belongs to a class ofcytotoxic agents selected from the group consisting of tubulinstabilizers, tubulin destabilizers, anti-metabolites, purine synthesisinhibitors, nucleoside analogs, DNA alkylating agents, DNA modifyingagents, and vascular disrupting agents. 28) The method of claim 26,wherein the cytotoxic agent is used alone or in combinations of one ormore cytotoxic agents selected from the group consisting ofcalicheamycin, doxorubicin, taxol, methotrexate, gemcitabine,cytarabine, vinblastin, daunorubicin, docetaxel, irinotecan, epothiloneB, epothilone D, cisplatin, carboplatin, and 5-fluoro-U. 29) A method ofreducing IFP in a solid tumor comprising the step of administering atherapeutically effective amount of an aptamer according to any ofclaims 1 through
 12. 30) A method of reducing IFP in a solid tumorcomprising the step of administering a therapeutically effective amountof a composition according to any of claims 13, 14 and
 18. 31) Themethod of claim 30, wherein the cytotoxic agent belongs to a class ofcytotoxic agents selected from the group consisting of tubulinstabilizers, tubulin destabilizers, anti-metabolites, purine synthesisinhibitors, nucleoside analogs, DNA alkylating agents, DNA modifyingagents, and vascular disrupting agents. 32) The method of claim 30,wherein the cytotoxic agent is used alone or in combinations of one ormore cytotoxic agents selected from the group consisting ofcalicheamycin, doxorubicin, taxol, methotrexate, gemcitabine,cytarabine, vinblastin, daunorubicin, docetaxel, irinotecan, epothiloneB, epothilone D, cisplatin, carboplatin, and 5-fluoro-U. 33) A method ofincreasing the permeability of a solid tumor to cytotoxic agentscomprising the step of administering a therapeutically effective amountof an aptamer according to any of claims 1 through
 12. 34) A method ofincreasing permeability of a solid tumor to cytotoxic agents comprisingthe step of administering a therapeutically effective amount of acomposition according to any of claims 13, 14 and
 18. 35) The method ofclaim 34, wherein the cytotoxic agent belongs to a class of cytotoxicagents selected from the group consisting of tubulin stabilizers,tubulin destabilizers, anti-metabolites, purine synthesis inhibitors,nucleoside analogs, DNA alkylating agents, DNA modifying agents, andvascular disrupting agents. 36) The method of claim 34, wherein thecytotoxic agent is used alone or in combinations of one or morecytotoxic agents selected from the group consisting of calicheamycin,doxorubicin, taxol, methotrexate, gemcitabine, cytarabine, vinblastin,daunorubicin, docetaxel, irinotecan, epothilone B, epothilone D,cisplatin, carboplatin, and 5-fluoro-U. 37) A method of reducingconstitutive expression of platelet derived growth factor in a tumorcomprising the step of administering a therapeutically effective amountof an aptamer according to any of claims 1 through
 12. 38) A method ofreducing constitutive activation of platelet derived growth factor in atumor comprising the step of administering a therapeutically effectiveamount of a composition according to any of claims 13, 14 and
 18. 39)The method of claim 38, wherein the cytotoxic agent belongs to a classof cytotoxic agents selected from the group consisting of tubulinstabilizers, tubulin destabilizers, anti-metabolites, purine synthesisinhibitors, nucleoside analogs, DNA alkylating agents, DNA modifyingagents, and vascular disrupting agents. 40) The method of claim 38,wherein the cytotoxic agent is used alone or in combinations of one ormore cytotoxic agents selected from the group consisting ofcalicheamycin, doxorubicin, taxol, methotrexate, gemcitabine,cytarabine, vinblastin, daunorubicin, docetaxel, irinotecan, epothiloneB, epothilone D, cisplatin, carboplatin, and 5-fluoro-U. 41) A method ofreducing angiogenesis and neovascularization in a solid tumor comprisingthe step of administering a therapeutically effective amount of anaptamer according to any of claims 1 through
 12. 42) A method ofreducing angiogenesis and neovascularization in a solid tumor comprisingthe step of administering a therapeutically effective amount of acomposition according to any of claims 13, 14 and
 18. 43) The method ofclaim 42, wherein the cytotoxic agent belongs to a class of cytotoxicagents selected from the group consisting of tubulin stabilizers,tubulin destabilizers, anti-metabolites, purine synthesis inhibitors,nucleoside analogs, DNA alkylating, and DNA modifying agents. 44) Themethod of claim 42, wherein the cytotoxic agent is used alone or incombinations of one or more cytotoxic agents selected from the groupconsisting of calicheamycin, doxorubicin, taxol, methotrexate,gemcitabine, cytarabine, vinblastin, daunorubicin, docetaxel,irinotecan, epothilone B, epothilone D, cisplatin, carboplatin, and5-fluoro-U. 45) The method of any one of claims 21 through 44 whereinsaid cancer or tumor is PDGF mediated cancer or tumor. 46) The method ofany one of claims 21 through 44 wherein said PDGF mediated cancer ortumor is selected from the group consisting of glioblastomas, chronicmyelomonocytic leukemia, dermafibrosarcoma protuberans, gastrointestinalstromal tumors and soft tissue sarcomas.